WO2016033327A1

WO2016033327A1 - E. coli-based production of beta-lactamase

Info

Publication number: WO2016033327A1
Application number: PCT/US2015/047187
Authority: WO
Inventors: Andrew BRISTOL; Michael Kaleko
Original assignee: Synthetic Biologics, Inc.
Priority date: 2014-08-28
Filing date: 2015-08-27
Publication date: 2016-03-03
Also published as: US11034966B2; CA2958755C; EP3186379A4; JP6803328B2; JP2017529833A; US20210332373A1; US20180030458A1; US20230279409A1; CN106574273A; CN106574273B; EP3186379A1; AU2015308897B2; CA2958755A1; US11542510B2; AU2015308897A1; EP3186379B1

Abstract

The invention relates to, in part, improved methods for the production of beta-lactamase using Escherichia coli (E. coli) cells. High yield production of beta-lactamase is achieved using methods of the invention.

Description

E. COLI-BASED PRODUCTION OF BETA- LACTAMASE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/043,360, filed August 28, 2014, the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to, in part, improved methods for the production of beta-lactamases using Escherichia coli (E. coli) cells. High yield production of beta-lactamase, including those suitable for pharmaceutical formulations, is achieved using methods of the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: SYN-005PC-SequenceListing.txt; date recorded: August 20, 2015; file size: 19 KB).

BACKGROUND

Beta-lactam antibiotics are characterized by a beta-lactam ring in their molecular structure. The integrity of the beta-lactam ring is essential for the biological activity, which results in the inactivation of a set of transpeptidases that catalyze the final cross-linking reactions of peptidoglycan synthesis. Members of the beta-lactam antibiotics family include penicillins, cephalosporins, clavams (or oxapenams), cephamycins and carbapenems.

Beta-lactamases are bacterial defensive enzymes that hydrolyze beta-lactam antibiotics. Gram-negative bacteria produce beta-lactamases to achieve resistance to beta-lactam antibiotics. Particularly, beta-lactamases are able to efficiently catalyze the irreversible hydrolysis of the amide bond of the beta-lactam ring resulting in biologically inactive product(s).

Humans may be considered to be a "superorganism" which is a conglomerate of mammalian and microbial cells, with the latter estimated to outnumber the former by ten to one. This microbial component, and its microbial genetic repertoire, the microbiome, is roughly 100-times greater than that of the human host. Strikingly, despite this enormous diversity of foreign organisms, the human immune system generally maintains a state of synergy. This is particularly true of the distal Gl tract, which houses up to 1000 distinct bacterial species and an estimated excess of 1 x10¹⁴ microorganisms, and appears to be central in defining human host health status. Loss of the careful balance in the microbiome, especially in the Gl tract, can lead to various diseases. Antibiotic medical treatments, which are needed to treat certain aspects of disease, can induce disruption in the microbiome, including in the Gl tract, and lead to further disease. For instance, certain parentally administered beta-lactams like ampicillin, ceftriaxone, cefoperazone, and piperacillin are, in part, eliminated via biliary excretion into the proximal part of the small intestine (duodenum). Residual unabsorbed beta-lactams in the intestinal tract may cause an undesirable effect on the ecological balance of normal intestinal microbiota resulting in, for example, Clostridium difficile infection (CDI), antibiotic-associated diarrhea, overgrowth of pathogenic bacteria such as vancomycin resistant enterococci (VRE), extended-spectrum beta-lactamase producing Gram- negative bacilli (ESBL), and fungi, and selection of antibiotic-resistance strains among both normal intestinal microbiota and potential pathogen bacteria.

One approach for avoiding or rebalancing the ecological balance of normal intestinal microbiota is the therapeutic use of beta-lactamases, for example, by inactivating excreted or unabsorbed antibiotics in the Gl tract, thereby maintaining a normal intestinal microbiota and preventing its overgrowth with potentially pathogenic microorganisms.

Accordingly, there is remains a need for efficient methods of producing beta-lactamases at a commercial scale for use in therapeutic intervention.

SUMMARY OF THE INVENTION

The present invention provides an improved method for the production of a beta-lactamase polypeptide in Escherichia coii (E. coii) cells. The method includes providing a host £ coii cell transformed with a vector comprising a sequence encoding the beta-lactamase polypeptide. The £ coii cell is cultured to induce expression of the beta-lactamase in the cytoplasm. Soluble fractions are subsequently prepared from the £. coii cell to recover the beta-lactase polypeptide.

The methods of the invention allows for production of beta-lactamases at a high yield. In an embodiment, the method yields at least 10 grams of the beta-lactamase polypeptide per liter of culture. In another embodiment, the method yields at least 15 grams of the beta-lactamase polypeptide per liter of culture.

Various strains of £ coii cells may be employed for the instant invention. For example, the £ coii cell may be selected from BL21 (DE3) or W3110. The beta-lactamase polypeptide is predominantly expressed in the cytoplasm of the £ coii cell. In certain embodiments, expression of the polypeptide is induced by adding isopropylthiogalactoside (IPTG) to the culture.

The disclosed method may be utilized to produce beta-lactamases and derivatives thereof. In one embodiment, the beta-lactamase polypeptide comprises a sequence having at least 60% identity with P1A. In another embodiment, the beta-lactamase polypeptide comprises a sequence having at least 60% identity with P2A. In yet another embodiment, the beta-lactamase polypeptide comprises a sequence having at least 60% identity with P3A. In a further embodiment, the beta-lactamase polypeptide comprises a sequence having at least 60% identity with P4A. In various embodiments, the present methods are used to produce beta-lactamases useful for microbiome-protecting therapy.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows a multi-fermenter computer system (MFCS) CLD977 fermentation plot of batch age (hours) vs. airflow (AIRFL (l/min), second line from top), temperature (TEMP (°C), top line), stirring rate (STIRR (RPM), second line from the bottom), pH (third line), and percent oxygen (PO2, bottom line).

Fig. 2 shows a MFCS CLD990 fermentation plot of batch age (hours) vs. airflow (AIRFL (l/min), second line from top), temperature (TEMP (°C), top line), stirring rate (STIRR (RPM), second line from the bottom), pH (third line), and percent oxygen (PO2, bottom line).

Fig. 3 shows a MFCS fermentation exit gas analysis plot of batch age (hours) vs. CLD977 (3/13C039) and CLD990 (4/13C040) oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) (mM/l/hr). Labeled from left to right, the first line corresponds to CLD977 OUR, the second line corresponds to CLD977 CER, the third line corresponds to CLD990 OUR and the fourth line corresponds to CLD990 CER.

Fig. 4 shows a biomass plot for CLD977 (3/13C039) and CLD990 (4/13C040) of batch time (hours) vs. OD₆₀o and dry cell weight (DCW (g/L)). CLD977 OD600 and DCW lines correspond to the top line and second from bottom line, respectively. CLD990 OD₆oo and DCW lines correspond to the second from top line and bottom line, respectively.

Fig. 5 shows bacterial gram stains for CLD977 and CLD990 at the end of batch phase and after fermentation is complete (final sample).

Fig. 6 shows SDS PAGE analysis of CLD977 (3/13C039) time course samples from pre-induction to the end of fermentation compared to control standards.

Fig. 7 shows SDS PAGE analysis of CLD990 (4/13C040) time course samples from pre-induction to the end of fermentation compared to control standards.

Fig. 8 shows SDS PAGE analysis of sonicated samples from CLD977 (3/13C039) and CLD990 (4/13C040) compared to control standards. CLD977 and CLD990 yielded mostly soluble protein. Only faint product bands are seen for the insoluble fraction.

Fig. 9 shows a standard curve of Time (sec) vs. Absorbance for Controls 1 and 2 as well as reference standard dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L. Controls 1 and 2 were preset dilutions of 1.0 μg/mL ran as duplicates. Fig. 10 shows a standard end point curve of Standard Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec. Specifically, enzymatic reaction was measured at time = 60 sec. The absorbance was measured at time = 0 sec which was then subtracted from the 60 sec measurement. Several dilutions of the reference standard were tested to generate a standard curve.

Fig. 11 shows a standard curve of Time (sec) vs. Absorbance for CLD981 (3/13C037 (also referred to as 37)) at 12 hours, 24 hours, 48 hours, as well as the periplasmic osmotic shock fraction (OS2). Specifically, OS2 is the second buffer fraction prepared from an E. coli pellet and represents the periplasmic space fraction.

Fig. 12 shows a standard end point curve of Time (sec) vs. Absorbance for CLD981 (3/13C037) OS1 samples.

Fig. 13 shows a standard curve of Time (sec) vs. Absorbance for CLD982 (4/13C038 (also referred to as 38)) 12h, 24h, 48h, and OS1 and OS2 48h post induction.

Fig. 14 shows a standard curve of Time (sec) vs. Absorbance for Control 1 and 2 (combined into control standard) as well as reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L.

Fig. 15 shows a standard end point curve of Standard Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec.

Fig. 16 shows a standard curve of Time (sec) vs. Absorbance for CLD981 (37) and CLD982 (38) OS1 and OS2 48h post induction. Table 3 is a summary of assay plate 2 activity and titer results for CLD981 and CLD982 OS1 and OS2 along with controls 1 and 2.

Fig. 17 shows a standard curve of Time (sec) vs. Absorbance for Control 1 and 2 (combined as control standard) as well as reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L.

Fig. 18 shows a standard end point curve of Standard Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec.

Fig. 19 shows a standard curve of Time (sec) vs. Absorbance for CLD977 (39) and CLD 990 (40) for both the second to last and last time point post induction (unlabeled = sonication) as well as the last time point post induction (Bug buster).

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discovery that a beta-lactamase polypeptide can be overproduced in high yields in £ coli cells. Specifically, high yield production is achieved by expressing the polypeptide in the cytoplasm of £ coli cells and subsequently recovering the polypeptide from soluble fractions prepared from the cells. Prior to the present invention, it was well established that beta-lactamases, such as the beta-lactamase from Bacillus licheniformis, are mostly found in the cell envelope and periplasmic fractions of £ coli cells. See Mezes, ei a/., J Biol Chem (1983), 258(18): 11211-11218. Particularly, beta-lactamase from Bacillus licheniformis is found to be completely absent in the cytoplasm. Id.

Further still, production of beta-lactamases from £ coli cells has generally been inefficient leading to an overall yield on the scale of milligrams of the enzyme per liter of culture. See, for example, Shaw ei a/., Protein Expr Purif. (1991), 2(2-3): 151-157. Given that the beta-lactamses are from Bacillus licheniformis, it is expected that production of these enzymes in Bacillus strains may provide a higher yield. However, studies shown herein demonstrate that even when produced in Bacillus subtilis cells, the yield of beta-lactamases is low. Accordingly, it is surprising that the present invention achieves an overall yield of beta-lactamases on the scale of grams per liter of culture.

Accordingly, the present invention provides an improved method for the production of a beta-lactamase polypeptide in Escherichia coli (E. coli) cells. The method includes providing a host £ coli cell transformed with a vector comprising a sequence encoding the beta-lactamase polypeptide. The £ coli cell is cultured to induce expression of the beta-lactamase in the cytoplasm. Soluble fractions are subsequently prepared from the £ coli cell for recovery of the beta-lactase polypeptide.

The present invention allows for high-yield production of a beta-lactamase polypeptide in £ coli cells. In various embodiments, methods of the present invention provides a yield of at least about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 16 grams, about 17 grams, about 18 grams, about 19 grams, about 20 grams, about 22 grams, about 24 grams, about 26 grams, about 28 grams, about 30 grams, about 35 grams, about 40 grams, about 45 grams, or about 50 grams of the beta-lactamase polypeptide per liter of culture. In one embodiment, at least about 10 grams of the beta- lactase polypeptide per liter of culture is recovered. In another embodiment, about at least 15 grams of the beta- lactase polypeptide per liter of culture is recovered. In a further embodiment, at least about 18 grams of the beta- lactase polypeptide per liter of culture is recovered.

In various embodiments, the present methods provide one or more of greater yield and improved purity as compared to a 6ac/7/us-based expression system such as, for example, those described in US Patent No. 7,319,030, the entire contents of which are hereby incorporated by reference. In various embodiments, the present methods provide one or more of greater yield and improved purity as compared to a method for producing a desired polypeptide product using a non-sporulating Bacillus subtilis strain, in which a deletion region of at least 150 nucleotides has been deleted from its sigG gene, the method involving transforming the strain with a polynucleotide construct encoding a recombinant polypeptide, expressing the polynucleotide construct, and recovering the recombinant polypeptide. In some embodiments the method comprises deleting at least part of either of the two functional regions of the sigG gene (i.e. the regions which code for amino acids 67 to 80 or 229 to 248).

In various embodiments, the present methods provide about a 5-fold, or about a 7.5-fold, or about a 10-fold, or about a 15-fold improvement in yield in E. coli versus a 6ac/7/us-based expression system such as, for example, those described in US Patent No. 7,319,030.

Various E. coli cell can be used with the present invention. Illustrative E. coli cells include, but are not limited to, BL21 (DES), W3110, DH5a, HMS174, and derivatives thereof. In one embodiment, the E. coli cell is the BL21 (DES) strain. In another embodiment, the E. coli cell is W3110 strain. The genotype of W3110 is E coli K12 F-, λ- , IN (rrnD-rrnE)l , rph-1. It is a Gram negative, rod-shaped, facultative anaerobe, and its genealogy is well described (Bachmann, BJ 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol.Rev. 36(4):525-57). There have been no modifications of this strain prior to transformation with the B3214 plasmid.

The present invention is used to produce beta-lactamase polypeptides at a high yield. In various aspects, the beta-lactamases polypeptide has the sequence of SEQ ID NO: 1 (Bacillus licheniformis PenP, i.e., P1A) or is derived by one or more mutations of SEQ ID NO: 1. Provided herein is the 263 amino acid sequence of the P1A enzyme (after removal of a 31 amino acid signal sequence and the QASKT (Gln-Ala-Ser-Lys-Thr) pentapeptide at the N terminus, see SEQ ID NO: 3). As described herein, mutations may be made to this sequence to generate beta-lactamase derivatives that may be produced by methods of the invention.

SEQ ID NO: 1

EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQR

ITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEV

TNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIR

AGVPDGWEVADKTGAASYGTRNDIAIIWPPKGDPWLAVLSSRDKKDAKYDDKLIAEATKWM

KALNMNGK.

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 1.

In some embodiments, SEQ ID NO: 1 may have a Met and/or Thr preceding the first residue of the sequence. In various embodiments, the Met may be cleaved. As described herein, mutations may be made to the sequence comprising the Met and/or Thr preceding the first residue to generate beta-lactamase derivatives. Also provided herein is the 299 amino acid sequence of the P1A enzyme before removal of a 31 amino acid signal sequence and the QASKT (Gln-Ala-Ser-Lys-Thr) pentapeptide at the N terminus as SEQ ID NO: 3:

SEQ ID NO: 3

MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAQASKTEMKDDFAKLEEQFDAKLGIFALDTGTN RTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLK ELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTST ARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVADKTGAASYGTR NDIAIIWPPKGDPWLAVLSSRDKKDAKYDDKLIAEATKVVMKALNMNGK

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 3.

Further, the beta-lactamase polypeptide may include additional upstream residues from the first residue of SEQ ID NO: 1 (see, e.g., JBC 258 (18): 11211 , 1983, the contents of which are hereby incorporated by reference- including the exo-large and exo-small versions of penP and penP1). Further, the beta-lactamase polypeptide may also include additional downstream residues from the last residue of SEQ ID NO: 1.

The polynucleotide sequence of P1A (after removal of a 31 amino acid signal sequence and the QAKST pentapeptide at the N terminus) is provided as SEQ ID NO: 2. As described herein, mutations may be made to this sequence to generate the beta-lactamase derivatives (including, taking into account degeneracy of the genetic code).

SEQ ID NO: 2

gagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacg gtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaag atctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacg ctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagttt gaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtga aactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcg agcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgata aaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatc cagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaa cggcaaataa

In some embodiments, the polynucleotide of the present invention has at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 2.

Also provided is the polynucleotide sequence of P1A before the removal of a 31 amino acid signal sequence and the QASKT pentapeptide at the N terminus as SEQ ID NO: 4. As described herein, mutations may be made to this sequence to generate beta-lactamase derivatives (including, taking into account degeneracy of the genetic code).

SEQ ID NO: 4

atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcag cgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatac aggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgca acagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcac gttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaa ttggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaat gaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaactt ccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacgg ttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctg tcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgat

aaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the polynucleotide of the present invention has at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 4.

In some embodiments, mutagenesis of a beta-lactamase (e.g. a class A beta-lactamase) is performed to derive advantageous enzymes (e.g. those that can target a broad spectra of antibiotics). In some embodiments, beta- lactamases derivatives are obtained by site-directed mutagenesis, random mutagenesis, and/or directed evolution approaches. In some embodiments, mutation design is based on, inter alia, structural data (e.g. crystal structure data, homolog models, etc.) of the following: P1A crystal structure (Knox and Moews, J. Mol Biol., 220, 435^55 (1991)), CTX-M-44 (1 BZA (Ibuka ef al. Journal of Molecular Biology Volume 285, Issue 5 2079-2087 (1999), 1 IYS (Ibuka et al. Biochemistry, 2003, 42 (36): 10634-43), 1 IYO, 1 IYP and 1 IYQ (Shimamura et al. 2002 J. Biol. Chem. 277:46601-08), Proteus vulgaris K1 (1 HZO, Nugaka ef al. J Mol Biol. 2002 Mar 15;317(1 ): 109-17) and Proteus penneri HugA (Liassine ef a/. Antimicrob Agents Chemother. 2002 Jan;46(1):216-9. 2002), and reviewed in Bonnet, Antimicrob. Agents Chemother 48(1): 1-14 (2004) (for CTM-X), the contents of all of these documents are hereby incorporated by reference in their entirety). In some embodiments, the present mutations are informed by analysis of structural data (e.g. crystal structure data, homolog models, efc.) of any one of the following beta-lactamases: P1A (see, e.g. US Patent No. 5,607,671 , the contents of which are hereby incorporated by reference), P2A (see, e.g., WO 2007/147945, the contents of which are hereby incorporated by reference), P3A (see, e.g., WO 2011/148041 , the contents of which are hereby incorporated by reference), CTX- M-3, CTX-M-4, CTX-M-5, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15, CTX-M-16, CTX-M-18, CTX-M-19, CTX- M-25, CTX-M-26, CTX-M-27, CTX-M-32, CTX-M-44, CTX-M-45, and CTX-M-54. Such information is available to one skilled in the art at known databases, for example, Swiss-Prot Protein Sequence Data Bank, NCBI, and PDB.

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention includes one or more (e.g. about 1 , or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about

80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150) mutations relative to SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence with at least 30, 35, 40, 50, 51 , 52, 53, 54, 55, 56, 57,

58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87,

88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3 (or about 60%, about 65%, about 70%, or about 75%, or about 80%, or about 85%, or about 90, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 3). In various embodiments, one or more amino acid of SEQ ID NO: 1 or SEQ ID NO: 3 is substituted with a naturally occurring amino acid, such as a hydrophilic amino acid (e.g. a polar and positively charged hydrophilic amino acid, such as arginine (R) or lysine (K); a polar and neutral of charge hydrophilic amino acid, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic amino acid, such as aspartate (D) or glutamate (E), or an aromatic, polar and positively charged hydrophilic amino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. a hydrophobic, aliphatic amino acid such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V), a hydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan (W), or tyrosine (Y) or a non-classical amino acid (e.g. selenocysteine, pyrrolysine, N-formylmethionine β-alanine, GABA and δ-Aminolevulinic acid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β methyl amino acids, C a -methyl amino acids, N a -methyl amino acids, and amino acid analogs in general).

In illustrative embodiments, inventive mutations include, but are not limited to one or more (e.g. about 1 , or about

2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150) of the following mutations to SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence with at least 30, 35, 40, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94,

95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3 (or about 70%, or about 75%, or about 80%, or about 85%, or about 90, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 3): GlulAla; Glul Cys; GlulAsp; Glul Phe; GlulGly; Glul His; Glul lle;

Metl Lys; Glul Leu; Glul Met; GlulAsn; Glul Pro; Glul GIn; GlulArg; Glul Ser; GlulThr; Glu al; GlulTrp;

GlulTyr; Met2Ala; Met2Cys; Met2Asp; Met2Glu; Met2Phe; Met2Gly; Met2His; Met2lle; Metl Lys; Met2Leu;

Met2Asn; Met2Pro; Met2Gln; Met2Arg; Met2Ser; Met2Thr; Met2Val; Met2Trp; Met2Tyr; Lys3Ala; Lys3Cys;

Lys3Asp; Lys3Glu; Lys3Phe; Lys3Gly; Lys3His; Lys3lle; Lys3Leu; Lys3Met; Lys3Asn; Lys3Pro; Lys3Gln;

Lys3Arg; Lys3Ser; Lys3Thr; Lys3Val; Lys3Trp; Lys3Tyr; Asp4Ala; Asp4Cys; Asp4Glu; Asp4Phe; Asp4Gly;

Asp4His; Asp4lle; Asp4Lys; Asp4Leu; Asp4Met; Asp4Asn; Asp4Pro; Asp4Gln; Asp4Arg; Asp4Ser; Asp4Thr;

Asp4Val; Asp4Trp; Asp4Tyr; Asp5Ala; Asp5Cys; Asp5Glu; Asp5Phe; Asp5Gly; Asp5His; Asp5lle; Asp5Lys;

Asp5Leu; Asp5Met; Asp5Asn; Asp5Pro; Asp5Gln; Asp5Arg; Asp5Ser; Asp5Thr; Asp5Val; Asp5Trp; Asp5Tyr;

Phe6Ala; Phe6Cys; Phe6Asp; Phe6Glu; Phe6Gly; Phe6His; Phe6lle; Phe6Lys; Phe6Leu; Phe6Met; Phe6Asn;

Phe6Pro; Phe6Gln; Phe6Arg; Phe6Ser; Phe6Thr; Phe6Val; Phe6Trp; Phe6Tyr; Ala7Cys; Ala7Asp; Ala7Glu;

Ala7Phe; Ala7Gly; Ala7His; Ala7lle; Ala7Lys; Ala7Leu; Ala7Met; Ala7Asn; Ala7Pro; Ala7Gln; Ala7Arg; Ala7Ser;

Ala7Thr; Ala7Val; Ala7Trp; Ala7Tyr; Lys8Ala; Lys8Cys; Lys8Asp; Lys8Glu; Lys8Phe; Lys8Gly; Lys8His; Lys8lle;

Lys8Leu; Lys8Met; Lys8Asn; Lys8Pro; Lys8Gln; Lys8Arg; Lys8Ser; Lys8Thr; Lys8Val; Lys8Trp; Lys8Tyr;

Leu9Ala; Leu9Cys; Leu9Asp; Leu9Glu; Leu9Phe; Leu9Gly; Leu9His; Leu9lle; Leu9Lys; Leu9Met; Leu9Asn;

Leu9Pro; Leu9Gln; Leu9Arg; Leu9Ser; Leu9Thr; Leu9Val; Leu9Trp; Leu9Tyr; Glul OAIa; Glul OCys; Glul OAsp;

Glul OPhe; Glul OGly; Glul OHis; Glul Olle; Glul OLys; Glul OLeu; Glul OMet; Glul OAsn; Glul OPro; Glul OGIn;

Glul OArg; Glul OSer; Glul OThr; Glul OVal; Glul OTrp; Glul OTyr; Glu11Ala; Glu11 Cys; Glu11Asp; Glu11 Phe;

Glu11 Gly; Glu11 His; Glu11 lle; Glu11 Lys; Glu11 Leu; Glu11 Met; Glu11Asn; Glu11 Pro; Glu11 Gln; Glu11Arg;

Glu11 Ser; Glu11Thr; Glu11Val; Glu11Trp; Glu11Tyr; Gln12Ala; Gln12Cys; Gln12Asp; Gln12Glu; Gln12Phe;

Gln12Gly; Gln12His; Gln12lle; Gln12Lys; Gln12Leu; Gln12Met; Gln12Asn; Gln12Pro; Gln12Arg; Gln12Ser;

Gln12Thr; Gln12Val; Gln12Trp; Gln12Tyr; Phe13Ala; Phe13Cys; Phe13Asp; Phe13Glu; Phe13Gly; Phe13His;

Phe13lle; Phe13Lys; Phe13Leu; Phe13Met; Phe13Asn; Phe13Pro; Phe13Gln; Phe13Arg; Phe13Ser; Phe13Thr; Phe13Val; Phe13Trp; Phe13Tyr; Asp14Ala; Asp14Cys; Asp14Glu; Asp14Phe; Asp14Gly; Asp14His; Asp14lle; Asp14Lys; Asp14Leu; Asp14Met; Asp14Asn; Asp14Pro; Asp14Gln; Asp14Arg; Asp14Ser; Asp14Thr; Asp14Val; Asp14Trp; Asp14Tyr; Ala15Cys; Ala15Asp; Ala15Glu; Ala15Phe; Ala15Gly; Ala15His; Ala15lle; Ala15Lys; Ala15Leu; Ala15Met; Ala15Asn; Ala15Pro; Ala15Gln; Ala15Arg; Ala15Ser; Ala15Thr; Ala15Val; Ala15Trp; Ala15Tyr; Lys16Ala; Lys16Cys; Lys16Asp; Lys16Glu; Lys16Phe; Lys16Gly; Lys16His; Lys16lle; Lys16Leu; Lys16Met; Lys16Asn; Lys16Pro; Lys16Gln; Lys16Arg; Lys16Ser; Lys16Thr; Lys16Val; Lys16Trp; Lys16Tyr; Leu17Ala; Leu17Cys; Leu17Asp; Leu17Glu; Leu17Phe; Leu17Gly; Leu17His; Leu17lle; Leu17Lys; Leu17Met; Leu17Asn; Leu17Pro; Leu17Gln; Leu17Arg; Leu17Ser; Leu17Thr; Leu17Val; Leu17Trp; Leu17Tyr; Gly18Ala; Gly18Cys; Gly18Asp; Gly18Glu; Gly18Phe; Gly18His; Gly18lle; Gly18Lys; Gly18Leu; Gly18Met; Gly18Asn; Gly18Pro; Gly18Gln; Gly18Arg; Gly18Ser; Gly18Thr; Gly18Val; Gly18Trp; Gly18Tyr; He19Ala; He19Cys; He19Asp; He19Glu; He19Phe; He19Gly; He19His; He19Lys; He19Leu; He19Met; He19Asn; He19Pro; He19Gln; He19Arg; He19Ser; He19Thr; He19Val; He19Trp; He19Tyr; Phe20Ala; Phe20Cys; Phe20Asp; Phe20Glu; Phe20Gly; Phe20His; Phe20lle; Phe20Lys; Phe20Leu; Phe20Met; Phe20Asn; Phe20Pro; Phe20Gln; Phe20Arg; Phe20Ser; Phe20Thr; Phe20Val; Phe20Trp; Phe20Tyr; Ala21Cys; Ala21Asp; Ala21Glu; Ala21 Phe; Ala21Gly; Ala21 His; Ala21lle; Ala21Lys; Ala21Leu; Ala21 Met; Ala21Asn; Ala21 Pro; Ala21Gln; Ala21Arg; Ala21Ser; Ala21Thr; Ala21Val; Ala21Trp; Ala21Tyr; Leu22Ala; Leu22Cys; Leu22Asp; Leu22Glu; Leu22Phe; Leu22Gly; Leu22His; Leu22lle; Leu22Lys; Leu22Met; Leu22Asn; Leu22Pro; Leu22Gln; Leu22Arg; Leu22Ser; Leu22Thr; Leu22Val; Leu22Trp; Leu22Tyr; Asp23Ala; Asp23Cys; Asp23Glu; Asp23Phe; Asp23Gly; Asp23His; Asp23lle; Asp23Lys; Asp23Leu; Asp23Met; Asp23Asn; Asp23Pro; Asp23Gln; Asp23Arg; Asp23Ser; Asp23Thr; Asp23Val; Asp23Trp; Asp23Tyr; Thr24Ala; Thr24Cys; Thr24Asp; Thr24Glu; Thr24Phe; Thr24Gly; Thr24His; Thr24lle; Thr24Lys; Thr24Leu; Thr24Met; Thr24Asn; Thr24Pro; Thr24Gln; Thr24Arg; Thr24Ser; Thr24Val; Thr24Trp; Thr24Tyr; Gly25Ala; Gly25Cys; Gly25Asp; Gly25Glu; Gly25Phe; Gly25His; Gly25lle; Gly25Lys; Gly25Leu; Gly25Met; Gly25Asn; Gly25Pro; Gly25Gln; Gly25Arg; Gly25Ser; Gly25Thr; Gly25Val; Gly25Trp; Gly25Tyr; Thr26Ala; Thr26Cys; Thr26Asp; Thr26Glu; Thr26Phe; Thr26Gly; Thr26His; Thr26lle; Thr26Lys; Thr26Leu; Thr26Met; Thr26Asn; Thr26Pro; Thr26Gln; Thr26Arg; Thr26Ser; Thr26Val; Thr26Trp; Thr26Tyr; Asn27Ala; Asn27Cys; Asn27Asp; Asn27Glu; Asn27Phe; Asn27Gly; Asn27His; Asn27lle; Asn27Lys; Asn27Leu; Asn27Met; Asn27Pro; Asn27Gln; Asn27Arg; Asn27Ser; Asn27Thr; Asn27Val; Asn27Trp; Asn27Tyr; Arg28Ala; Arg28Cys; Arg28Asp; Arg28Glu; Arg28Phe; Arg28Gly; Arg28His; Arg28lle; Arg28Lys; Arg28Leu; Arg28Met; Arg28Asn; Arg28Pro; Arg28Gln; Arg28Ser; Arg28Thr; Arg28Val; Arg28Trp; Arg28Tyr; Thr29Ala; Thr29Cys; Thr29Asp; Thr29Glu; Thr29Phe; Thr29Gly; Thr29His; Thr29lle; Thr29Lys; Thr29Leu; Thr29Met; Thr29Asn; Thr29Pro; Thr29Gln; Thr29Arg; Thr29Ser; Thr29Val; Thr29Trp; Thr29Tyr; Val30Ala; Val30Cys; Val30Asp; Val30Glu; Val30Phe; Val30Gly; Val30His; Val30lle; Val30Lys; Val30Leu; Val30Met; Val30Asn; Val30Pro; Val30Gln; Val30Arg; Val30Ser; Val30Thr; Val30Trp; Val30Tyr; Ala31Ala; Ala31Cys; Ala31Asp; Ala31Glu; Ala31 Phe; Ala31Gly; Ala31 His; Ala31 lle; Ala31Lys; Ala31Leu; Ala31 Met; Ala31Asn; Ala31 Pro; Ala31Gln; Ala31Arg; Ala31Ser; Ala31Thr; Ala31Val; Ala31Trp; Ala31Tyr; Tyr32Ala; Tyr32Cys; Tyr32Asp; Tyr32Glu; Tyr32Phe; Tyr32Gly; Tyr32His; Tyr32lle; Tyr32Lys; Tyr32Leu; Tyr32Met; Tyr32Asn; Tyr32Pro; Tyr32Gln; Tyr32Arg; Tyr32Ser; Tyr32Thr; Tyr32Val; Tyr32Trp; Arg33Ala; Arg33Cys; Arg33Asp; Arg33Glu; Arg33Phe; Arg33Gly; Arg33His; Arg33lle; Arg33Lys; Arg33Leu; Arg33Met; Arg33Asn; Arg33Pro; Arg33Gln; Arg33Ser; Arg33Thr; Arg33Val; Arg33Trp; Arg33Tyr; Pro34Ala; Pro34Cys; Pro34Asp; Pro34Glu; Pro34Phe; Pro34Gly; Pro34His; Pro34lle; Pro34Lys; Pro34Leu; Pro34Met; Pro34Asn; Pro34Gln; Pro34Arg; Pro34Ser; Pro34Thr; Pro34Val; Pro34Trp; Pro34Tyr; Asp35Ala; Asp35Cys; Asp35Glu; Asp35Phe; Asp35Gly; Asp35His; Asp35lle; Asp35Lys; Asp35Leu; Asp35Met; Asp35Asn; Asp35Pro; Asp35Gln; Asp35Arg; Asp35Ser; Asp35Thr; Asp35Val; Asp35Trp; Asp35Tyr; Glu36Ala; Glu36Cys; Glu36Asp; Glu36Phe; Glu36Gly; Glu36His; Glu36lle; Glu36Lys; Glu36Leu; Glu36Met; Glu36Asn; Glu36Pro; Glu36Gln; Glu36Arg; Glu36Ser; Glu36Thr; Glu36Val; Glu36Trp; Glu36Tyr; Arg37Ala; Arg37Cys; Arg37Asp; Arg37Glu; Arg37Phe; Arg37Gly; Arg37His; Arg37lle; Arg37Lys; Arg37Leu; Arg37Met; Arg37Asn; Arg37Pro; Arg37Gln; Arg37Ser; Arg37Thr; Arg37Val; Arg37Trp; Arg37Tyr; Phe38Ala; Phe38Cys; Phe38Asp; Phe38Glu; Phe38Gly; Phe38His; Phe38lle; Phe38Lys; Phe38Leu; Phe38Met; Phe38Asn; Phe38Pro; Phe38Gln; Phe38Arg; Phe38Ser; Phe38Thr; Phe38Val; Phe38Trp; Phe38Tyr; Ala39Cys; Ala39Asp; Ala39Glu; Ala39Phe; Ala39Gly; Ala39His; Ala39lle; Ala39Lys; Ala39Leu; Ala39Met; Ala39Asn; Ala39Pro; Ala39Gln; Ala39Arg; Ala39Ser; Ala39Thr; Ala39Val; Ala39Trp; Ala39Tyr; Phe40Ala; Phe40Cys; Phe40Asp; Phe40Glu; Phe40Gly; Phe40His; Phe40lle; Phe40Lys; Phe40Leu; Phe40Met; Phe40Asn; Phe40Pro; Phe40Gln; Phe40Arg; Phe40Ser; Phe40Thr; Phe40Val; Phe40Trp; Phe40Tyr; Ala41 Cys; Ala41Asp; Ala41 Glu; Ala41 Phe; Ala41 Gly; Ala41 His; Ala41 lle; Ala41 Lys; Ala41 Leu; Ala41 Met; Ala41Asn; Ala41 Pro; Ala41 Gln; Ala41Arg; Ala41 Ser; Ala41 Thr; Ala41Val; Ala41Trp; Ala41 Tyr; Ser42Ala; Ser42Cys; Ser42Asp; Ser42Glu; Ser42Phe; Ser42Gly; Ser42His; Ser42lle; Ser42Lys; Ser42Leu; Ser42Met; Ser42Asn; Ser42Pro; Ser42Gln; Ser42Arg; Ser42Thr; Ser42Val; Ser42Trp; Ser42Tyr; Thr43Ala; Thr43Cys; Thr43Asp; Thr43Glu; Thr43Phe; Thr43Gly; Thr43His; Thr43lle; Thr43Lys; Thr43Leu; Thr43Met; Thr43Asn; Thr43Pro; Thr43Gln; Thr43Arg; Thr43Ser; Thr43Val; Thr43Trp; Thr43Tyr; He44Ala; He44Cys; He44Asp; He44Glu; He44Phe; He44Gly; He44His; He44Lys; He44Leu; He44Met; He44Asn; He44Pro; He44Gln; He44Arg; He44Ser; He44Thr; He44Val; He44Trp; He44Tyr; Lys45Ala; Lys45Cys; Lys45Asp; Lys45Glu; Lys45Phe; Lys45Gly; Lys45His; Lys45lle; Lys45Leu; Lys45Met; Lys45Asn; Lys45Pro; Lys45Gln; Lys45Arg; Lys45Ser; Lys45Thr; Lys45Val; Lys45Trp; Lys45Tyr; Ala46Cys; Ala46Asp; Ala46Glu; Ala46Phe; Ala46Gly; Ala46His; Ala46lle; Ala46Lys; Ala46Leu; Ala46Met; Ala46Asn; Ala46Pro; Ala46Gln; Ala46Arg; Ala46Ser; Ala46Thr; Ala46Val; Ala46Trp; Ala46Tyr; Leu47Ala; Leu47Cys; Leu47Asp; Leu47Glu; Leu47Phe; Leu47Gly; Leu47His; Leu47lle; Leu47Lys; Leu47Met; Leu47Asn; Leu47Pro; Leu47Gln; Leu47Arg; Leu47Ser; Leu47Thr; Leu47Val; Leu47Trp; Leu47Tyr; Thr48Ala; Thr48Cys; Thr48Asp; Thr48Glu; Thr48Phe; Thr48Gly; Thr48His; Thr48lle; Thr48Lys; Thr48Leu; Thr48Met; Thr48Asn; Thr48Pro; Thr48Gln; Thr48Arg; Thr48Ser; Thr48Val; Thr48Trp; Thr48Tyr; Val49Ala; Val49Cys; Val49Asp; Val49Glu; Val49Phe; Val49Gly; Val49His; Val49lle; Val49Lys; Val49Leu; Val49Met; Val49Asn; Val49Pro; Val49Gln; Val49Arg; Val49Ser; Val49Thr; Val49Trp; Val49Tyr; Gly50Ala; GlySOCys; GlySOAsp; Gly50Glu; Gly50Phe; Gly50His; Gly50lle; GlySOLys; Gly50Leu; GlySOMet; Gly50Asn; GlySOPro; Gly50Gln; GlySOArg; Gly50Ser; Gly50Thr; Gly50Val; Gly50Trp; GlySOTyr; Val51Ala; Val51 Cys; Val51Asp; Val51 Glu; Val51 Phe; Val51 Gly; Val51 His; Val51 lle; Val51 Lys; Val51 Leu; Val51 Met; Val51Asn; Val51 Pro; Val51 Gln; ValSIArg; Val51 Ser; Val51 Thr; Val51 Trp; Val51Tyr; Leu52Ala; Leu52Cys; Leu52Asp; Leu52Glu; Leu52Phe; Leu52Gly; Leu52His; Leu52lle; Leu52Lys; Leu52Met; Leu52Asn; Leu52Pro; Leu52Gln; Leu52Arg; Leu52Ser; Leu52Thr; Leu52Val; Leu52Trp; Leu52Tyr; Leu53Ala; Leu53Cys; Leu53Asp; Leu53Glu; Leu53Phe; Leu53Gly; Leu53His; Leu53lle; Leu53Lys; Leu53Met; Leu53Asn; Leu53Pro; Leu53Gln; Leu53Arg; Leu53Ser; Leu53Thr; Leu53Val; Leu53Trp; Leu53Tyr; Gln54Ala; Gln54Cys; Gln54Asp; Gln54Glu; Gln54Phe; Gln54Gly; Gln54His; Gln54lle; Gln54Lys; Gln54Leu; Gln54Met; Gln54Asn; Gln54Pro; Gln54Arg; Gln54Ser; Gln54Thr; Gln54Val; Gln54Trp; Gln54Tyr; Gln55Ala; Gln55Cys; Gln55Asp; Gln55Glu; Gln55Phe; Gln55Gly; Gln55His; Gln55lle; Gln55Lys; Gln55Leu; Gln55Met; Gln55Asn; Gln55Pro; Gln55Arg; Gln55Ser; Gln55Thr; Gln55Val; Gln55Trp; Gln55Tyr; Lys56Ala; Lys56Cys; Lys56Asp; Lys56Glu; Lys56Phe; Lys56Gly; Lys56His; Lys56lle; Lys56Leu; Lys56Met; Lys56Asn; Lys56Pro; Lys56Gln; Lys56Arg; Lys56Ser; Lys56Thr; Lys56Val; Lys56Trp; Lys56Tyr; Ser57Ala; Ser57Cys; Ser57Asp; Ser57Glu; Ser57Phe; Ser57Gly; Ser57His; Ser57lle; Ser57Lys; Ser57Leu; Ser57Met; Ser57Asn; Ser57Pro; Ser57Gln; Ser57Arg; Ser57Thr; Ser57Val; Ser57Trp; Ser57Tyr; He58Ala; He58Cys; He58Asp; He58Glu; He58Phe; He58Gly; He58His; He58Lys; He58Leu; He58Met; He58Asn; He58Pro; He58Gln; He58Arg; He58Ser; He58Thr; He58Val; He58Trp; He58Tyr; Glu59Ala; Glu59Cys; Glu59Asp; Glu59Phe; Glu59Gly; Glu59His; Glu59lle; Glu59Lys; Glu59Leu; Glu59Met; Glu59Asn; Glu59Pro; Glu59Gln; Glu59Arg; Glu59Ser; Glu59Thr; Glu59Val; Glu59Trp; Glu59Tyr; Asp60Ala; Asp60Cys; Asp60Glu; Asp60Phe; Asp60Gly; Asp60His; Asp60lle; Asp60Lys; Asp60Leu; Asp60Met; Asp60Asn; Asp60Pro; Asp60Gln; Asp60Arg; Asp60Ser; Asp60Thr; Asp60Val; Asp60Trp; Asp60Tyr; Leu61Ala; Leu61 Cys; Leu61Asp; Leu61 Glu; Leu61 Phe; Leu61 Gly; Leu61 His; Leu61 lle; Leu61 Lys; Leu61 Met; Leu61Asn; Leu61 Pro; Leu61 Gln; Leu61Arg; Leu61 Ser; Leu61 Thr; Leu61Val; Leu61Trp; Leu61Tyr; Asn62Ala; Asn62Cys; Asn62Asp; Asn62Glu; Asn62Phe; Asn62Gly; Asn62His; Asn62lle; Asn62Lys; Asn62Leu; Asn62Met; Asn62Pro; Asn62Gln; Asn62Arg; Asn62Ser; Asn62Thr; Asn62Val; Asn62Trp; Asn62Tyr; Gln63Ala; Gln63Cys; Gln63Asp; Gln63Glu; Gln63Phe; Gln63Gly; Gln63His; Gln63lle; Gln63Lys; Gln63Leu; Gln63Met; Gln63Asn; Gln63Pro; Gln63Arg; Gln63Ser; Gln63Thr; Gln63Val; Gln63Trp; Gln63Tyr; Arg64Ala; Arg64Cys; Arg64Asp; Arg64Glu; Arg64Phe; Arg64Gly; Arg64His; Arg64lle; Arg64Lys; Arg64Leu; Arg64Met; Arg64Asn; Arg64Pro; Arg64Gln; Arg64Ser; Arg64Thr; Arg64Val; Arg64Trp; Arg64Tyr; He65Ala; He65Cys; He65Asp; He65Glu; He65Phe; He65Gly; He65His; He65Lys; He65Leu; He65Met; He65Asn; He65Pro; He65Gln; He65Arg; He65Ser; He65Thr; He65Val; He65Trp; He65Tyr; Thr66Ala; Thr66Cys; Thr66Asp; Thr66Glu; Thr66Phe; Thr66Gly; Thr66His; Thr66lle; Thr66Lys; Thr66Leu; Thr66Met; Thr66Asn; Thr66Pro; Thr66Gln; Thr66Arg; Thr66Ser; Thr66Val; Thr66Trp; Thr66Tyr; Tyr67Ala; Tyr67Cys; Tyr67Asp; Tyr67Glu; Tyr67Phe; Tyr67Gly; Tyr67His; Tyr67lle; Tyr67Lys; Tyr67Leu; Tyr67Met; Tyr67Asn; Tyr67Pro; Tyr67Gln; Tyr67Arg; Tyr67Ser; Tyr67Thr; Tyr67Val; Tyr67Trp; Thr68Ala; Thr68Cys; Thr68Asp; Thr68Glu; Thr68Phe; Thr68Gly; Thr68His; Thr68lle; Thr68Lys; Thr68Leu; Thr68Met; Thr68Asn; Thr68Pro; Thr68Gln; Thr68Arg; Thr68Ser; Thr68Val; Thr68Trp; Thr68Tyr; Arg69Ala; Arg69Cys; Arg69Asp; Arg69Glu; Arg69Phe; Arg69Gly; Arg69His; Arg69lle; Arg69Lys; Arg69Leu; Arg69Met; Arg69Asn; Arg69Pro; Arg69Gln; Arg69Ser; Arg69Thr; Arg69Val; Arg69Trp; Arg69Tyr; Asp70Ala; Asp70Cys; Asp70Glu; Asp70Phe; Asp70Gly; Asp70His; Asp70lle; Asp70Lys; Asp70Leu; Asp70Met; Asp70Asn; Asp70Pro; Asp70Gln; Asp70Arg; Asp70Ser; Asp70Thr; Asp70Val; Asp70Trp; Asp70Tyr; Asp71Ala; Asp71 Cys; Asp71 Glu; Asp71 Phe; Asp71 Gly; Asp71 His; Asp71 He; Asp71 Lys; Asp71 Leu; Asp71 Met; Asp71Asn; Asp71 Pro; Asp71 Gln; Asp71Arg; Asp71 Ser; Asp71 Thr; Asp71Val; Asp71 Trp; Asp71 Tyr; Leu72Ala; Leu72Cys; Leu72Asp; Leu72Glu; Leu72Phe; Leu72Gly; Leu72His; Leu72lle; Leu72Lys; Leu72Met; Leu72Asn; Leu72Pro; Leu72Gln; Leu72Arg; Leu72Ser; Leu72Thr; Leu72Val; Leu72Trp; Leu72Tyr; Val73Ala; Val73Cys; Val73Asp; Val73Glu; Val73Phe; Val73Gly; Val73His; Val73lle; Val73Lys; Val73Leu; Val73Met; Val73Asn; Val73Pro; Val73Gln; Val73Arg; Val73Ser; Val73Thr; Val73Trp; Val73Tyr; Asn74Ala; Asn74Cys; Asn74Asp; Asn74Glu; Asn74Phe; Asn74Gly; Asn74His; Asn74lle; Asn74Lys; Asn74Leu; Asn74Met; Asn74Pro; Asn74Gln; Asn74Arg; Asn74Ser; Asn74Thr; Asn74Val; Asn74Trp; Asn74Tyr; Tyr75Ala; Tyr75Cys; Tyr75Asp; Tyr75Glu; Tyr75Phe; Tyr75Gly; Tyr75His; Tyr75lle; Tyr75Lys; Tyr75Leu; Tyr75Met; Tyr75Asn; Tyr75Pro; Tyr75Gln; Tyr75Arg; Tyr75Ser; Tyr75Thr; Tyr75Val; Tyr75Trp; Asn76Ala; Asn76Cys; Asn76Asp; Asn76Glu; Asn76Phe; Asn76Gly; Asn76His; Asn76lle; Asn76Lys; Asn76Leu; Asn76Met; Asn76Pro; Asn76Gln; Asn76Arg; Asn76Ser; Asn76Thr; Asn76Val; Asn76Trp; Asn76Tyr; Pro77Ala; Pro77Cys; Pro77Asp; Pro77Glu; Pro77Phe; Pro77Gly; Pro77His; Pro77lle; Pro77Lys; Pro77Leu; Pro77Met; Pro77Asn; Pro77Gln; Pro77Arg; Pro77Ser; Pro77Thr; Pro77Val; Pro77Trp; Pro77Tyr; He78Ala; He78Cys; He78Asp; He78Glu; He78Phe; He78Gly; He78His; He78Lys; He78Leu; He78Met; He78Asn; He78Pro; He78Gln; He78Arg; He78Ser; He78Thr; He78Val; He78Trp; He78Tyr; Thr79Ala; Thr79Cys; Thr79Asp; Thr79Glu; Thr79Phe; Thr79Gly; Thr79His; Thr79lle; Thr79Lys; Thr79Leu; Thr79Met; Thr79Asn; Thr79Pro; Thr79Gln; Thr79Arg; Thr79Ser; Thr79Val; Thr79Trp; Thr79Tyr; Glu80Ala; Glu80Cys; Glu80Asp; Glu80Phe; Glu80Gly; Glu80His; Glu80lle; Glu80Lys; Glu80Leu; Glu80Met; Glu80Asn; Glu80Pro; Glu80Gln; Glu80Arg; Glu80Ser; Glu80Thr; Glu80Val; Glu80Trp; Glu80Tyr; Lys81Ala; Lys81 Cys; Lys81Asp; Lys81 Glu; Lys81 Phe; Lys81 Gly; Lys81 His; Lys81 lle; Lys81 Leu; Lys81 Met; Lys81Asn; Lys81 Pro; Lys81 Gln; Lys81Arg; Lys81 Ser; Lys81 Thr; Lys81Val; Lys81Trp; Lys81 Tyr; His82Ala; His82Cys; His82Asp; His82Glu; His82Phe; His82Gly; His82lle; His82Lys; His82Leu; His82Met; His82Asn; His82Pro; His82Gln; His82Arg; His82Ser; His82Thr; His82Val; His82Trp; His82Tyr; Val83Ala; Val83Cys; Val83Asp; Val83Glu; Val83Phe; Val83Gly; Val83His; Val83lle; Val83Lys; Val83Leu; Val83Met; Val83Asn; Val83Pro; Val83Gln; Val83Arg; Val83Ser; Val83Thr; Val83Trp; Val83Tyr; Asp84Ala; Asp84Cys; Asp84Glu; Asp84Phe; Asp84Gly; Asp84His; Asp84lle; Asp84Lys; Asp84Leu; Asp84Met; Asp84Asn; Asp84Pro; Asp84Gln; Asp84Arg; Asp84Ser; Asp84Thr; Asp84Val; Asp84Trp; Asp84Tyr; Thr85Ala; Thr85Cys; Thr85Asp; Thr85Glu; Thr85Phe; Thr85Gly; Thr85His; Thr85lle; Thr85Lys; Thr85Leu; Thr85Met; Thr85Asn; Thr85Pro; Thr85Gln; Thr85Arg; Thr85Ser; Thr85Val; Thr85Trp; Thr85Tyr; Gly86Ala; Gly86Cys; Gly86Asp; Gly86Glu; Gly86Phe; Gly86His; Gly86lle; Gly86Lys; Gly86Leu; Gly86Met; Gly86Asn; Gly86Pro; Gly86Gln; Gly86Arg; Gly86Ser; Gly86Thr; Gly86Val; Gly86Trp; Gly86Tyr; Met87Ala; Met87Cys; Met87Asp; Met87Glu; Met87Phe; Met87Gly; Met87His; Met87lle; Met87Lys; Met87Leu; Met87Asn; Met87Pro; Met87Gln; Met87Arg; Met87Ser; Met87Thr; Met87Val; Met87Trp; Met87Tyr; Thr88Ala; Thr88Cys; Thr88Asp; Thr88Glu; Thr88Phe; Thr88Gly; Thr88His; Thr88lle; Thr88Lys; Thr88Leu; Thr88Met; Thr88Asn; Thr88Pro; Thr88Gln; Thr88Arg; Thr88Ser; Thr88Val; Thr88Trp; Thr88Tyr; Leu89Ala; Leu89Cys; Leu89Asp; Leu89Glu; Leu89Phe; Leu89Gly; Leu89His; Leu89lle; Leu89Lys; Leu89Met; Leu89Asn; Leu89Pro; Leu89Gln; Leu89Arg; Leu89Ser; Leu89Thr; Leu89Val; Leu89Trp; Leu89Tyr; Lys90Ala; Lys90Cys; Lys90Asp; Lys90Glu; Lys90Phe; Lys90Gly; Lys90His; Lys90lle; Lys90Leu; Lys90Met; Lys90Asn; Lys90Pro; Lys90Gln; Lys90Arg; Lys90Ser; Lys90Thr; Lys90Val; Lys90Trp; Lys90Tyr; Glu91Ala; Glu91 Cys; Glu91Asp; Glu91 Phe; Glu91 Gly; Glu91 His; Glu91 lie; Glu91 Lys; Glu91 Leu; Glu91 Met; Glu91Asn; Glu91 Pro; Glu91 Gln; Glu91Arg; Glu91 Ser; Glu91Thr; Glu91Val; Glu91Trp; Glu91Tyr; Leu92Ala; Leu92Cys; Leu92Asp; Leu92Glu; Leu92Phe; Leu92Gly; Leu92His; Leu92lle; Leu92Lys; Leu92Met; Leu92Asn; Leu92Pro; Leu92Gln; Leu92Arg; Leu92Ser; Leu92Thr; Leu92Val; Leu92Trp; Leu92Tyr; Ala93Cys; Ala93Asp; Ala93Glu; Ala93Phe; Ala93Gly; Ala93His; Ala93lle; Ala93Lys; Ala93Leu; Ala93Met; Ala93Asn; Ala93Pro; Ala93Gln; Ala93Arg; Ala93Ser; Ala93Thr; Ala93Val; Ala93Trp; Ala93Tyr; Asp94Ala; Asp94Cys; Asp94Glu; Asp94Phe; Asp94Gly; Asp94His; Asp94lle; Asp94Lys; Asp94Leu; Asp94Met; Asp94Asn; Asp94Pro; Asp94Gln; Asp94Arg; Asp94Ser; Asp94Thr; Asp94Val; Asp94Trp; Asp94Tyr; Ala95Cys; Ala95Asp; Ala95Glu; Ala95Phe; Ala95Gly; Ala95His; Ala95lle; Ala95Lys; Ala95Leu; Ala95Met; Ala95Asn; Ala95Pro; Ala95Gln; Ala95Arg; Ala95Ser; Ala95Thr; Ala95Val; Ala95Trp; Ala95Tyr; Ser96Ala; Ser96Cys; Ser96Asp; Ser96Glu; Ser96Phe; Ser96Gly; Ser96His; Ser96lle; Ser96Lys; Ser96Leu; Ser96Met; Ser96Asn; Ser96Pro; Ser96Gln; Ser96Arg; Ser96Thr; Ser96Val; Ser96Trp; Ser96Tyr; Leu97Ala; Leu97Cys; Leu97Asp; Leu97Glu; Leu97Phe; Leu97Gly; Leu97His; Leu97lle; Leu97Lys; Leu97Met; Leu97Asn; Leu97Pro; Leu97Gln; Leu97Arg; Leu97Ser; Leu97Thr; Leu97Val; Leu97Trp; Leu97Tyr; Arg98Ala; Arg98Cys; Arg98Asp; Arg98Glu; Arg98Phe; Arg98Gly; Arg98His; Arg98lle; Arg98Lys; Arg98Leu; Arg98Met; Arg98Asn; Arg98Pro; Arg98Gln; Arg98Ser; Arg98Thr; Arg98Val; Arg98Trp; Arg98Tyr; Tyr99Ala; Tyr99Cys; Tyr99Asp; Tyr99Glu; Tyr99Phe; Tyr99Gly; Tyr99His; Tyr99lle; Tyr99Lys; Tyr99Leu; Tyr99Met; Tyr99Asn; Tyr99Pro; Tyr99Gln; Tyr99Arg; Tyr99Ser; Tyr99Thr; Tyr99Val; Tyr99Trp; Serl OOAIa; Serl OOCys; Serl OOAsp; SerlOOGIu; SerlOOPhe; Serl OOGly; SerlOOHis; Serl OOIIe; Serl OOLys; Serl OOLeu; SerlOOMet; Serl OOAsn; Serl OOPro; SerlOOGIn; Serl OOArg; Serl OOThr; Serl OOVal; Serl OOTrp; Serl OOTyr; Asp101Ala; Asp101 Cys; Asp101 Glu; Asp101 Phe; Asp101 Gly; Asp101 His; Asp101 lie; Asp101 Lys; Asp101 Leu; Asp101 Met; Asp101Asn; Asp101 Pro; Asp101 Gln; Asp101Arg; Asp101 Ser; Asp101Thr; Asp101Val; Asp101Trp; Asp101Tyr; Asn102Ala; Asn102Cys; Asn102Asp; Asn102Glu; Asn102Phe; Asn102Gly; Asn102His; Asn102lle; Asn102Lys; Asn102Leu; Asn102Met; Asn102Pro; Asn102Gln; Asn102Arg; Asn102Ser; Asn102Thr; Asn102Val; Asn102Trp; Asn102Tyr; Ala103Cys; Ala103Asp; Ala103Glu; Ala103Phe; Ala103Gly; Ala103His; Ala103lle; Ala103Lys; Ala103Leu; Ala103Met; Ala103Asn; Ala103Pro; Ala103Gln; Ala103Arg; Ala103Ser; Ala103Thr; Ala103Val; Ala103Trp; Ala103Tyr; Ala104Cys; Ala104Asp; Ala104Glu; Ala104Phe; Ala104Gly; Ala104His; Ala104lle; Ala104Lys; Ala104Leu; Ala104Met; Ala104Asn; Ala104Pro; Ala104Gln; Ala104Arg; Ala104Ser; Ala104Thr; Ala104Val; Ala104Trp; Ala104Tyr; Gln105Ala; Gln105Cys; Gln105Asp; Gln105Glu; Gln105Phe; Gln105Gly; Gln105His; Gln105lle; Gln105Lys; Gln105Leu; Gln105Met; Gln105Asn; Gln105Pro; Gln105Arg; Gln105Ser; Gln105Thr; Gln105Val; Gln105Trp; Gln105Tyr; Asn106Ala; Asn106Cys; Asn106Asp; Asn106Glu; Asn106Phe; Asn106Gly; Asn106His; Asn106lle; Asn106Lys; Asn106Leu; Asn106Met; Asn106Pro; Asn106Gln; Asn106Arg; Asn106Ser; Asn106Thr; Asn106Val; Asn106Trp; Asn106Tyr; Leu107Ala; Leu107Cys; Leu107Asp; Leu107Glu; Leu107Phe; Leu107Gly; Leu107His; Leu107lle; Leu107Lys; Leu107Met; Leu107Asn; Leu107Pro; Leu107Gln; Leu107Arg; Leu107Ser; Leu107Thr; Leu107Val; Leu107Trp; Leu107Tyr; He108Ala; He108Cys; He108Asp; He108Glu; He108Phe; He108Gly; He108His; He108Lys; He108Leu; He108Met; He108Asn; He108Pro; He108Gln; He108Arg; He108Ser; He108Thr; He108Val; He108Trp; He108Tyr; Leu109Ala; Leu109Cys; Leu109Asp; Leu109Glu; Leu109Phe; Leu109Gly; Leu109His; Leu109lle; Leu109Lys; Leu109Met; Leu109Asn; Leu109Pro; Leu109Gln; Leu109Arg; Leu109Ser; Leu109Thr; Leu109Val; Leu109Trp; Leu109Tyr; Lys110Ala; Lys110Cys; Lys110Asp; Lys110Glu; Lys110Phe; Lys110Gly; Lys110His; Lys110lle; Lys110Leu; Lys110Met; Lys110Asn; Lys110Pro; Lys110Gln; Lys110Arg; Lys110Ser; Lys110Thr; Lys110Val; Lys110Trp; Lys110Tyr; Gln111Ala; Gln111Cys; Gln111Asp; Gln111Glu; Gln111Phe; Gln111Gly; Gln111His; Gln111lle; Gln111Lys; Gln111Leu; Gln111Met; Gln111Asn; Gln111Pro; Gln111Arg; Gln111Ser; Gln111Thr; Gln111Val; Gln111Trp; Gln111Tyr; He112Ala; He112Cys; He112Asp; He112Glu; He112Phe; He112Gly; He112His; He112Lys; He112Leu; He112Met; He112Asn; He112Pro; He112Gln; He112Arg; He112Ser; He112Thr; He112Val; He112Trp; He112Tyr; Gly113Ala; Gly113Cys; Gly113Asp; Gly113Glu; Gly113Phe; Gly113His; Gly113lle; Gly113Lys; Gly113Leu; Gly113Met; Gly113Asn; Gly113Pro; Gly113Gln; Gly113Arg; Gly113Ser; Gly113Thr; Gly113Val; Gly113Trp; Gly113Tyr; Gly114Ala; Gly114Cys; Gly114Asp; Gly114Glu; Gly114Phe; Gly114His; Gly114lle; Gly114Lys; Gly114Leu; Gly114Met; Gly114Asn; Gly114Pro; Gly114Gln; Gly114Arg; Gly114Ser; Gly114Thr; Gly114Val; Gly114Trp; Gly114Tyr; Pro115Ala; Pro115Cys; Pro115Asp; Pro115Glu; Pro115Phe; Pro115Gly; Pro115His; Pro115lle; Pro115Lys; Pro115Leu; Pro115Met; Pro115Asn; Pro115Gln; Pro115Arg; Pro115Ser; Pro115Thr; Pro115Val; Pro115Trp; Pro115Tyr; Glu116Ala; Glu116Cys; Glu116Asp; Glu116Phe; Glu116Gly; Glu116His; Glu116lle; Glu116Lys; Glu116Leu; Glu116Met; Glu116Asn; Glu116Pro; Glu116Gln; Glu116Arg; Glu116Ser; Glu116Thr; Glu116Val; Glu116Trp; Glu116Tyr; Ser117Ala; Ser117Cys; Ser117Asp; Ser117Glu; Ser117Phe; Ser117Gly; Ser117His; Ser117lle; Ser117Lys; Ser117Leu; Ser117Met; Ser117Asn; Ser117Pro; Ser117Gln; Ser117Arg; Ser117Thr; Ser117Val; Ser117Trp; Ser117Tyr; Leu118Ala; Leu118Cys; Leu118Asp; Leu118Glu; Leu118Phe; Leu118Gly; Leu118His; Leu118lle; Leu118Lys; Leu118Met; Leu118Asn; Leu118Pro; Leu118Gln; Leu118Arg; Leu118Ser; Leu118Thr; Leu118Val; Leu118Trp; Leu118Tyr; Lys119Ala; Lys119Cys; Lys119Asp; Lys119Glu; Lys119Phe; Lys119Gly; Lys119His; Lys119lle; Lys119Leu; Lys119Met; Lys119Asn; Lys119Pro; Lys119Gln; Lys119Arg; Lys119Ser; Lys119Thr; Lys119Val; Lys119Trp; Lys119Tyr; Lys120Ala; Lys120Cys; Lys120Asp; Lys120Glu; Lys120Phe; Lys120Gly; Lys120His; Lys120lle; Lys120Leu; Lys120Met; Lys120Asn; Lys120Pro; Lys120Gln; Lys120Arg; Lys120Ser; Lys120Thr; Lys120Val; Lys120Trp; Lys120Tyr; Glu121Ala; Glu121Cys; Glu121Asp; Glu121Phe; Glu121Gly; Glu121His; Glu121lle; Glu121Lys; Glu121Leu; Glu121Met; Glu121Asn; Glu121Pro; Glu121Gln; Glu121Arg; Glu121Ser; Glu121Thr; Glu121Val; Glu121Trp; Glu121Tyr; Leu122Ala; Leu122Cys; Leu122Asp; Leu122Glu; Leu122Phe; Leu122Gly; Leu122His; Leu122lle; Leu122Lys; Leu122Met; Leu122Asn; Leu122Pro; Leu122Gln; Leu122Arg; Leu122Ser; Leu122Thr; Leu122Val; Leu122Trp; Leu122Tyr; Arg123Ala; Arg123Cys; Arg123Asp; Arg123Glu; Arg123Phe; Arg123Gly; Arg123His; Arg123lle; Arg123Lys; Arg123Leu; Arg123Met; Arg123Asn; Arg123Pro; Arg123Gln; Arg123Ser; Arg123Thr; Arg123Val; Arg123Trp; Arg123Tyr; Lys124Ala; Lys124Cys; Lys124Asp; Lys124Glu; Lys124Phe; Lys124Gly; Lys124His; Lys124lle; Lys124Leu; Lys124Met; Lys124Asn; Lys124Pro; Lys124Gln; Lys124Arg; Lys124Ser; Lys124Thr; Lys124Val; Lys124Trp; Lys124Tyr; He125Ala; He125Cys; He125Asp; He125Glu; He125Phe; He125Gly; He125His; He125Lys; He125Leu; He125Met; He125Asn; He125Pro; He125Gln; He125Arg; He125Ser; He125Thr; He125Val; He125Trp; He125Tyr; Gly126Ala; Gly126Cys; Gly126Asp; Gly126Glu; Gly126Phe; Gly126His; Gly126lle; Gly126Lys; Gly126Leu; Gly126Met; Gly126Asn; Gly126Pro; Gly126Gln; Gly126Arg; Gly126Ser; Gly126Thr; Gly126Val; Gly126Trp; Gly126Tyr; Asp127Ala; Asp127Cys; Asp127Glu; Asp127Phe; Asp127Gly; Asp127His; Asp127lle; Asp127Lys; Asp127Leu; Asp127Met; Asp127Asn; Asp127Pro; Asp127Gln; Asp127Arg; Asp127Ser; Asp127Thr; Asp127Val; Asp127Trp; Asp127Tyr; Glu128Ala; Glu128Cys; Glu128Asp; Glu128Phe; Glu128Gly; Glu128His; Glu128lle; Glu128Lys; Glu128Leu; Glu128Met; Glu128Asn; Glu128Pro; Glu128Gln; Glu128Arg; Glu128Ser; Glu128Thr; Glu128Val; Glu128Trp; Glu128Tyr; Val129Ala; Val129Cys; Val129Asp; Val129Glu; Val129Phe; Val129Gly; Val129His; Val129lle; Val129Lys; Val129Leu; Val129Met; Val129Asn; Val129Pro; Val129Gln; Val129Arg; Val129Ser; Val129Thr; Val129Trp; Val129Tyr; Thr130Ala; Thr130Cys; Thr130Asp; Thr130Glu; Thr130Phe; Thr130Gly; Thr130His; Thr130lle; Thr130Lys; Thr130Leu; Thr130Met; Thr130Asn; Thr130Pro; Thr130Gln; Thr130Arg; Thr130Ser; Thr130Val; Thr130Trp; Thr130Tyr; Asn131Ala; Asn131 Cys; Asn131Asp; Asn131Glu; Asn131 Phe; Asn131 Gly; Asn131 His; Asn131 lle; Asn131Lys; Asn131 Leu; Asn131 Met; Asn131 Pro; Asn131Gln; Asn131Arg; Asn131 Ser; Asn131Thr; Asn131Val; Asn131Trp; Asn131Tyr; Pro132Ala; Pro132Cys; Pro132Asp; Pro132Glu; Pro132Phe; Pro132Gly; Pro132His; Pro132lle; Pro132Lys; Pro132Leu; Pro132Met; Pro132Asn; Pro132Gln; Pro132Arg; Pro132Ser; Pro132Thr; Pro132Val; Pro132Trp; Pro132Tyr; Glu133Ala; Glu133Cys; Glu133Asp; Glu133Phe; Glu133Gly; Glu133His; Glu133lle; Glu133Lys; Glu133Leu; Glu133Met; Glu133Asn; Glu133Pro; Glu133Gln; Glu133Arg; Glu133Ser; Glu133Thr; Glu133Val; Glu133Trp; Glu133Tyr; Arg134Ala; Arg134Cys; Arg134Asp; Arg134Glu; Arg134Phe; Arg134Gly; Arg134His; Arg134lle; Arg134Lys; Arg134Leu; Arg134Met; Arg134Asn; Arg134Pro; Arg134Gln; Arg134Ser; Arg134Thr; Arg134Val; Arg134Trp; Arg134Tyr; Phe135Ala; Phe135Cys; Phe135Asp; Phe135Glu; Phe135Gly; Phe135His; Phe135lle; Phe135Lys; Phe135Leu; Phe135Met; Phe135Asn; Phe135Pro; Phe135Gln; Phe135Arg; Phe135Ser; Phe135Thr; Phe135Val; Phe135Trp; Phe135Tyr; Glu136Ala; Glu136Cys; Glu136Asp; Glu136Phe; Glu136Gly; Glu136His; Glu136lle; Glu136Lys; Glu136Leu; Glu136Met; Glu136Asn; Glu136Pro; Glu136Gln; Glu136Arg; Glu136Ser; Glu136Thr; Glu136Val; Glu136Trp; Glu136Tyr; Pro137Ala; Pro137Cys; Pro137Asp; Pro137Glu; Pro137Phe; Pro137Gly; Pro137His; Pro137lle; Pro137Lys; Pro137Leu; Pro137Met; Pro137Asn; Pro137Gln; Pro137Arg; Pro137Ser; Pro137Thr; Pro137Val; Pro137Trp; Pro137Tyr; Glu138Ala; Glu138Cys; Glu138Asp; Glu138Phe; Glu138Gly; Glu138His; Glu138lle; Glu138Lys; Glu138Leu; Glu138Met; Glu138Asn; Glu138Pro; Glu138Gln; Glu138Arg; Glu138Ser; Glu138Thr; Glu138Val; Glu138Trp; Glu138Tyr; Leu139Ala; Leu139Cys; Leu139Asp; Leu139Glu; Leu139Phe; Leu139Gly; Leu139His; Leu139lle; Leu139Lys; Leu139Met; Leu139Asn; Leu139Pro; Leu139Gln; Leu139Arg; Leu139Ser; Leu139Thr; Leu139Val; Leu139Trp; Leu139Tyr; Asn140Ala; Asn140Cys; Asn140Asp; Asn140Glu; Asn140Phe; Asn140Gly; Asn140His; Asn140lle; Asn140Lys; Asn140Leu; Asn140Met; Asn140Pro; Asn140Gln; Asn140Arg; Asn140Ser; Asn140Thr; Asn140Val; Asn140Trp; Asn140Tyr; Glu141Ala; Glu141Cys; Glu141Asp; Glu141 Phe; Glu141 Gly; Glu141 His; Glu141 lle; Glu141 Lys; Glu141 Leu; Glu141 Met; Glu141Asn; Glu141 Pro; Glu141 Gln; Glu141Arg; Glu141 Ser; Glu141Thr; Glu141Val; Glu141Trp; Glu141Tyr; Val142Ala; Val142Cys; Val142Asp; Val142Glu; Val142Phe; Val142Gly; Val142His; Val142lle; Val142Lys; Val142Leu; Val142Met; Val142Asn; Val 142Pro; Val142Gln; Val142Arg; Val142Ser; Val 142Thr; Val142Trp; Val142Tyr; Asn143Ala; Asn143Cys; Asn143Asp; Asn143Glu; Asn143Phe; Asn143Gly; Asn143His; Asn143lle; Asn143Lys; Asn143Leu; Asn143Met; Asn143Pro; Asn143Gln; Asn143Arg; Asn143Ser; Asn143Thr; Asn143Val; Asn143Trp; Asn143Tyr; Pro144Ala; Pro144Cys; Pro144Asp; Pro144Glu; Pro144Phe; Pro144Gly; Pro144His; Pro144lle; Pro144Lys; Pro144Leu; Pro144Met; Pro144Asn; Pro144Gln; Pro144Arg; Pro144Ser; Pro144Thr; Pro144Val; Pro144Trp; Pro144Tyr; Gly145Ala; Gly145Cys; Gly145Asp; Gly145Glu; Gly145Phe; Gly145His; Gly145lle; Gly145Lys; Gly145Leu; Gly145Met; Gly145Asn; Gly145Pro; Gly145Gln; Gly145Arg; Gly145Ser; Gly145Thr; Gly145Val; Gly145Trp; Gly145Tyr; Glu146Ala; Glu146Cys; Glu146Asp; Glu146Phe; Glu146Gly; Glu146His; Glu146lle; Glu146Lys; Glu146Leu; Glu146Met; Glu146Asn; Glu146Pro; Glu146Gln; Glu146Arg; Glu146Ser; Glu146Thr; Glu146Val; Glu146Trp; Glu146Tyr; Thr147Ala; Thr147Cys; Thr147Asp; Thr147Glu; Thr147Phe; Thr147Gly; Thr147His; Thr147lle; Thr147Lys; Thr147Leu; Thr147Met; Thr147Asn; Thr147Pro; Thr147Gln; Thr147Arg; Thr147Ser; Thr147Val; Thr147Trp; Thr147Tyr; Gln148Ala; Gln148Cys; Gln148Asp; Gln148Glu; Gln148Phe; Gln148Gly; Gln148His; Gln148lle; Gln148Lys; Gln148Leu; Gln148Met; Gln148Asn; Gln148Pro; Gln148Arg; Gln148Ser; Gln148Thr; Gln148Val; Gln148Trp; Gln148Tyr; Asp149Ala; Asp149Cys; Asp149Glu; Asp149Phe; Asp149Gly; Asp149His; Asp149lle; Asp149Lys; Asp149Leu; Asp149Met; Asp149Asn; Asp149Pro; Asp149Gln; Asp149Arg; Asp149Ser; Asp149Thr; Asp149Val; Asp149Trp; Asp149Tyr; Thr150Ala; Thr150Cys; Thr150Asp; Thr150Glu; Thr150Phe; Thr150Gly; Thr150His; Thr150lle; Thr150Lys; Thr150Leu; Thr150Met; Thr150Asn; Thr150Pro; Thr150Gln; Thr150Arg; Thr150Ser; Thr150Val; Thr150Trp; Thr150Tyr; Ser151Ala; Ser151 Cys; Ser151Asp; Ser151 Glu; Ser151 Phe; Ser151 Gly; Ser151 His; Ser151 lle; Ser151 Lys; Ser151 Leu; Ser151 Met; Ser151Asn; Ser151 Pro; Ser151 Gln; Ser151Arg; Ser151 Thr; Ser151Val; Ser151 Trp; Ser151Tyr; Thr152Ala; Thr152Cys; Thr152Asp; Thr152Glu; Thr152Phe; Thr152Gly; Thr152His; Thr152lle; Thr152Lys; Thr152Leu; Thr152Met; Thr152Asn; Thr152Pro; Thr152Gln; Thr152Arg; Thr152Ser; Thr152Val; Thr152Trp; Thr152Tyr; Ala153Cys; Ala153Asp; Ala153Glu; Ala153Phe; Ala153Gly; Ala153His; Ala153lle; Ala153Lys; Ala153Leu; Ala153Met; Ala153Asn; Ala153Pro; Ala153Gln; Ala153Arg; Ala153Ser; Ala153Thr; Ala153Val; Ala153Trp; Ala153Tyr; Arg154Ala; Arg154Cys; Arg154Asp; Arg154Glu; Arg154Phe; Arg154Gly; Arg154His; Arg154lle; Arg154Lys; Arg154Leu; Arg154Met; Arg154Asn; Arg154Pro; Arg154Gln; Arg154Ser; Arg154Thr; Arg154Val; Arg154Trp; Arg154Tyr; Ala155Cys; Ala155Asp; Ala155Glu; Ala155Phe; Ala155Gly; Ala155His; Ala155lle; Ala155Lys; Ala155Leu; Ala155Met; Ala155Asn; Ala155Pro; Ala155Gln; Ala155Arg; Ala155Ser; Ala155Thr; Ala155Val; Ala155Trp; Ala155Tyr; Leu156Ala; Leu156Cys; Leu156Asp; Leu156Glu; Leu156Phe; Leu156Gly; Leu156His; Leu156lle; Leu156Lys; Leu156Met; Leu156Asn; Leu156Pro; Leu156Gln; Leu156Arg; Leu156Ser; Leu156Thr; Leu156Val; Leu156Trp; Leu156Tyr; Val157Ala; Val157Cys; Val 157Asp; Val157Glu; Val157Phe; Val 157Gly; Val 157His; Val 157lle; Val157Lys; Val157Leu; Val 157Met; Val 157Asn; Val157Pro; Val157Gln; Val157Arg; Val157Ser; Val157Thr; Val157Trp; Val157Tyr; Thr158Ala; Thr158Cys; Thr158Asp; Thr158Glu; Thr158Phe; Thr158Gly; Thr158His; Thr158lle; Thr158Lys; Thr158Leu; Thr158Met; Thr158Asn; Thr158Pro; Thr158Gln; Thr158Arg; Thr158Ser; Thr158Val; Thr158Trp; Thr158Tyr; Ser159Ala; Ser159Cys; Ser159Asp; Ser159Glu; Ser159Phe; Ser159Gly; Ser159His; Ser159lle; Ser159Lys; Ser159Leu; Ser159Met; Ser159Asn; Ser159Pro; Ser159Gln; Ser159Arg; Ser159Thr; Ser159Val; Ser159Trp; Ser159Tyr; Leu160Ala; Leu160Cys; Leu160Asp; Leu160Glu; Leu160Phe; Leu160Gly; Leu160His; Leu160lle; Leu160Lys; Leu160Met; Leu160Asn; Leu160Pro; Leu160Gln; Leu160Arg; Leu160Ser; Leu160Thr; Leu160Val; Leu160Trp; Leu160Tyr; Arg161Ala; Arg161 Cys; Arg161Asp; Arg161 Glu; Arg161 Phe; Arg161Gly; Arg161 His; Arg161 lle; Arg161 Lys; Arg161 Leu; Arg161 Met; Arg161Asn; Arg161 Pro; Arg161 Gln; Arg161 Ser; Arg161Thr; Arg161Val; Arg161Trp; Arg161Tyr; Ala162Cys; Ala162Asp; Ala162Glu; Ala162Phe; Ala162Gly; Ala162His; Ala162lle; Ala162Lys; Ala162Leu; Ala162Met; Ala162Asn; Ala162Pro; Ala162Gln; Ala162Arg; Ala162Ser; Ala162Thr; Ala162Val; Ala162Trp; Ala162Tyr; Phe163Ala; Phe163Cys; Phe163Asp; Phe163Glu; Phe163Gly; Phe163His; Phe163lle; Phe163Lys; Phe163Leu; Phe163Met; Phe163Asn; Phe163Pro; Phe163Gln; Phe163Arg; Phe163Ser; Phe163Thr; Phe163Val; Phe163Trp; Phe163Tyr; Ala164Cys; Ala164Asp; Ala164Glu; Ala164Phe; Ala164Gly; Ala164His; Ala164lle; Ala164Lys; Ala164Leu; Ala164Met; Ala164Asn; Ala164Pro; Ala164Gln; Ala164Arg; Ala164Ser; Ala164Thr; Ala164Val; Ala164Trp; Ala164Tyr; Leu165Ala; Leu165Cys; Leu165Asp; Leu165Glu; Leu165Phe; Leu165Gly; Leu165His; Leu165lle; Leu165Lys; Leu165Met; Leu165Asn; Leu165Pro; Leu165Gln; Leu165Arg; Leu165Ser; Leu165Thr; Leu165Val; Leu165Trp; Leu165Tyr; Glu166Ala; Glu166Cys; Glu166Asp; Glu166Phe; Glu166Gly; Glu166His; Glu166lle; Glu166Lys; Glu166Leu; Glu166Met; Glu166Asn; Glu166Pro; Glu166Gln; Glu166Arg; Glu166Ser; Glu166Thr; Glu166Val; Glu166Trp; Glu166Tyr; Asp167Ala; Asp167Cys; Asp167Glu; Asp167Phe; Asp167Gly; Asp167His; Asp167lle; Asp167Lys; Asp167Leu; Asp167Met; Asp167Asn; Asp167Pro; Asp167Gln; Asp167Arg; Asp167Ser; Asp167Thr; Asp167Val; Asp167Trp; Asp167Tyr; Lys168Ala; Lys168Cys; Lys168Asp; Lys168Glu; Lys168Phe; Lys168Gly; Lys168His; Lys168lle; Lys168Leu; Lys168Met; Lys168Asn; Lys168Pro; Lys168Gln; Lys168Arg; Lys168Ser; Lys168Thr; Lys168Val; Lys168Trp; Lys168Tyr; Leu169Ala; Leu169Cys; Leu169Asp; Leu169Glu; Leu169Phe; Leu169Gly; Leu169His; Leu169lle; Leu169Lys; Leu169Met; Leu169Asn; Leu169Pro; Leu169Gln; Leu169Arg; Leu169Ser; Leu169Thr; Leu169Val; Leu169Trp; Leu169Tyr; Pro170Ala; Pro170Cys; Pro170Asp; Pro170Glu; Pro170Phe; Pro170Gly; Pro170His; Pro170lle; Pro170Lys; Pro170Leu; Pro170Met; Pro170Asn; Pro170Gln; Pro170Arg; Pro170Ser; Pro170Thr; Pro170Val; Pro170Trp; Pro170Tyr; Ser171Ala; Ser171 Cys; Ser171Asp; Ser171 Glu; Ser171 Phe; Ser171 Gly; Ser171 His; Ser171 lle; Ser171 Lys; Ser171Leu; Ser171 Met; Ser171Asn; Ser171 Pro; Ser171 Gln; Ser171Arg; Ser171Thr; Ser171Val; Ser171Trp; Ser171Tyr; Glu172Ala; Glu172Cys; Glu172Asp; Glu172Phe; Glu172Gly; Glu172His; Glu172lle; Glu172Lys; Glu172Leu; Glu172Met; Glu172Asn; Glu172Pro; Glu172Gln; Glu172Arg; Glu172Ser; Glu172Thr; Glu172Val; Glu172Trp; Glu172Tyr; Lys173Ala; Lys173Cys; Lys173Asp; Lys173Glu; Lys173Phe; Lys173Gly; Lys173His; Lys173lle; Lys173Leu; Lys173Met; Lys173Asn; Lys173Pro; Lys173Gln; Lys173Arg; Lys173Ser; Lys173Thr; Lys173Val; Lys173Trp; Lys173Tyr; Arg174Ala; Arg174Cys; Arg174Asp; Arg174Glu; Arg174Phe; Arg174Gly; Arg174His; Arg174lle; Arg174Lys; Arg174Leu; Arg174Met; Arg174Asn; Arg174Pro; Arg174Gln; Arg174Ser; Arg174Thr; Arg174Val; Arg174Trp; Arg174Tyr; Glu175Ala; Glu175Cys; Glu175Asp; Glu175Phe; Glu175Gly; Glu175His; Glu175lle; Glu175Lys; Glu175Leu; Glu175Met; Glu175Asn; Glu175Pro; Glu175Gln; Glu175Arg; Glu175Ser; Glu175Thr; Glu175Val; Glu175Trp; Glu175Tyr; Leu176Ala; Leu176Cys; Leu176Asp; Leu176Glu; Leu176Phe; Leu176Gly; Leu176His; Leu176lle; Leu176Lys; Leu176Met; Leu176Asn; Leu176Pro; Leu176Gln; Leu176Arg; Leu176Ser; Leu176Thr; Leu176Val; Leu176Trp; Leu176Tyr; Leu177Ala; Leu177Cys; Leu177Asp; Leu177Glu; Leu177Phe; Leu177Gly; Leu177His; Leu177lle; Leu177Lys; Leu177Met; Leu177Asn; Leu177Pro; Leu177Gln; Leu177Arg; Leu177Ser; Leu177Thr; Leu177Val; Leu177Trp; Leu177Tyr; He178Ala; He178Cys; He178Asp; He178Glu; He178Phe; He178Gly; He178His; He178Lys; He178Leu; He178Met; He178Asn; He178Pro; He178Gln; He178Arg; He178Ser; He178Thr; He178Val; He178Trp; He178Tyr; Asp179Ala; Asp179Cys; Asp179Glu; Asp179Phe; Asp179Gly; Asp179His; Asp179lle; Asp179Lys; Asp179Leu; Asp179Met; Asp179Asn; Asp179Pro; Asp179Gln; Asp179Arg; Asp179Ser; Asp179Thr; Asp179Val; Asp179Trp; Asp179Tyr; Trp180Ala; Trp180Cys; Trp180Asp; Trp180Glu; Trp180Phe; Trp180Gly; Trp180His; Trp180lle; Trp180Lys; Trp180Leu; Trp180Met; Trp180Asn; Trp180Pro; Trp180Gln; Trp180Arg; Trp180Ser; Trp180Thr; Trp180Val; Trp180Tyr; Met181Ala; Met181 Cys; Met181Asp; Met181 Glu; Met181 Phe; Met181 Gly; Met181 His; Met181 lle; Met181 Lys; Met181 Leu; Met181Asn; Met181 Pro; Met181 Gln; Met181Arg; Met181 Ser; Met181 Thr; Met181Val; Met181 Trp; Met181 Tyr; Lys182Ala; Lys182Cys; Lys182Asp; Lys182Glu; Lys182Phe; Lys182Gly; Lys182His; Lys182lle; Lys182Leu; Lys182Met; Lys182Asn; Lys182Pro; Lys182Gln; Lys182Arg; Lys182Ser; Lys182Thr; Lys182Val; Lys182Trp; Lys182Tyr; Arg183Ala; Arg183Cys; Arg183Asp; Arg183Glu; Arg183Phe; Arg183Gly; Arg183His; Arg183lle; Arg183Lys; Arg183Leu; Arg183Met; Arg183Asn; Arg183Pro; Arg183Gln; Arg183Ser; Arg183Thr; Arg183Val; Arg183Trp; Arg183Tyr; Asn184Ala; Asn184Cys; Asn184Asp; Asn184Glu; Asn184Phe; Asn184Gly; Asn184His; Asn184lle; Asn184Lys; Asn184Leu; Asn184Met; Asn184Pro; Asn184Gln; Asn184Arg; Asn184Ser; Asn184Thr; Asn184Val; Asn184Trp; Asn184Tyr; Thr185Ala; Thr185Cys; Thr185Asp; Thr185Glu; Thr185Phe; Thr185Gly; Thr185His; Thr185lle; Thr185Lys; Thr185Leu; Thr185Met; Thr185Asn; Thr185Pro; Thr185Gln; Thr185Arg; Thr185Ser; Thr185Val; Thr185Trp; Thr185Tyr; Thr186Ala; Thr186Cys; Thr186Asp; Thr186Glu; Thr186Phe; Thr186Gly; Thr186His; Thr186lle; Thr186Lys; Thr186Leu; Thr186Met; Thr186Asn; Thr186Pro; Thr186Gln; Thr186Arg; Thr186Ser; Thr186Val; Thr186Trp; Thr186Tyr; Gly187Ala; Gly187Cys; Gly187Asp; Gly187Glu; Gly187Phe; Gly187His; Gly187lle; Gly187Lys; Gly187Leu; Gly187Met; Gly187Asn; Gly187Pro; Gly187Gln; Gly187Arg; Gly187Ser; Gly187Thr; Gly187Val; Gly187Trp; Gly187Tyr; Asp188Ala; Asp188Cys; Asp188Glu; Asp188Phe; Asp188Gly; Asp188His; Asp188lle; Asp188Lys; Asp188Leu; Asp188Met; Asp188Asn; Asp188Pro; Asp188Gln; Asp188Arg; Asp188Ser; Asp188Thr; Asp188Val; Asp188Trp; Asp188Tyr; Ala189Cys; Ala189Asp; Ala189Glu; Ala189Phe; Ala189Gly; Ala189His; Ala189lle; Ala189Lys; Ala189Leu; Ala189Met; Ala189Asn; Ala189Pro; Ala189Gln; Ala189Arg; Ala189Ser; Ala189Thr; Ala189Val; Ala189Trp; Ala189Tyr; Leu190Ala; Leu190Cys; Leu190Asp; Leu190Glu; Leu190Phe; Leu190Gly; Leu190His; Leu190lle; Leu190Lys; Leu190Met; Leu190Asn; Leu190Pro; Leu190Gln; Leu190Arg; Leu190Ser; Leu190Thr; Leu190Val; Leu190Trp; Leu190Tyr; He191Ala; He191 Cys; He191Asp; He191 Glu; He191 Phe; He191 Gly; He191 His; He191 Lys; He191 Leu; He191 Met; He191Asn; He191 Pro; He191 Gln; He191Arg; He191 Ser; He191 Thr; He191Val; He191 Trp; He191Tyr; Arg192Ala; Arg192Cys; Arg192Asp; Arg192Glu; Arg192Phe; Arg192Gly; Arg192His; Arg192lle; Arg192Lys; Arg192Leu; Arg192Met; Arg192Asn; Arg192Pro; Arg192Gln; Arg192Ser; Arg192Thr; Arg192Val; Arg192Trp; Arg192Tyr; Ala193Cys; Ala193Asp; Ala193Glu; Ala193Phe; Ala193Gly; Ala193His; Ala193lle; Ala193Lys; Ala193Leu; Ala193Met; Ala193Asn; Ala193Pro; Ala193Gln; Ala193Arg; Ala193Ser; Ala193Thr; Ala193Val; Ala193Trp; Ala193Tyr; Gly194Ala; Gly194Cys; Gly194Asp; Gly194Glu; Gly194Phe; Gly194His; Gly194lle; Gly194Lys; Gly194Leu; Gly194Met; Gly194Asn; Gly194Pro; Gly194Gln; Gly194Arg; Gly194Ser; Gly194Thr; Gly194Val; Gly194Trp; Gly194Tyr; Val 195Ala; Val 195Cys; Val195Asp; Val 195Glu; Val 195Phe; Val195Gly; Val195His; Val195lle; Val195Lys; Val 195Leu; Val195Met; Val195Asn; Val 195Pro; Val 195Gln; Val195Arg; Val195Ser; Val 195Thr; Val 195Trp; Val195Tyr; Pro196Ala; Pro196Cys; Pro196Asp; Pro196Glu; Pro196Phe; Pro196Gly; Pro196His; Pro196lle; Pro196Lys; Pro196Leu; Pro196Met; Pro196Asn; Pro196Gln; Pro196Arg; Pro196Ser; Pro196Thr; Pro196Val; Pro196Trp; Pro196Tyr; Asp197Ala; Asp197Cys; Asp197Glu; Asp197Phe; Asp197Gly; Asp197His; Asp197lle; Asp197Lys; Asp197Leu; Asp197Met; Asp197Asn; Asp197Pro; Asp197Gln; Asp197Arg; Asp197Ser; Asp197Thr; Asp197Val; Asp197Trp; Asp197Tyr; Gly198Ala; Gly198Cys; Gly198Asp; Gly198Glu; Gly198Phe; Gly198His; Gly198lle; Gly198Lys; Gly198Leu; Gly198Met; Gly198Asn; Gly198Pro; Gly198Gln; Gly198Arg; Gly198Ser; Gly198Thr; Gly198Val; Gly198Trp; Gly198Tyr; Trp199Ala; Trp199Cys; Trp199Asp; Trp199Glu; Trp199Phe; Trp199Gly; Trp199His; Trp199lle; Trp199Lys; Trp199Leu; Trp199Met; Trp199Asn; Trp199Pro; Trp199Gln; Trp199Arg; Trp199Ser; Trp199Thr; Trp199Val; Trp199Tyr; Glu200Ala; Glu200Cys; Glu200Asp; Glu200Phe; Glu200Gly; Glu200His; Glu200lle; Glu200Lys; Glu200Leu; Glu200Met; Glu200Asn; Glu200Pro; Glu200Gln; Glu200Arg; Glu200Ser; Glu200Thr; Glu200Val; Glu200Trp; Glu200Tyr; Val201Ala; Val201 Cys; Val201Asp; Val201 Glu; Val201 Phe; Val201 Gly; Val201 His; Val201 lle; Val201 Lys; Val201 Leu; Val201 Met; Val201Asn; Val201 Pro; Val201 Gln; Val201Arg; Val201 Ser; Val201Thr; Val201 Trp; Val201Tyr; Ala202Cys; Ala202Asp; Ala202Glu; Ala202Phe; Ala202Gly; Ala202His; Ala202lle; Ala202Lys; Ala202Leu; Ala202Met; Ala202Asn; Ala202Pro; Ala202Gln; Ala202Arg; Ala202Ser; Ala202Thr; Ala202Val; Ala202Trp; Ala202Tyr; Asp203Ala; Asp203Cys; Asp203Glu; Asp203Phe; Asp203Gly; Asp203His; Asp203lle; Asp203Lys; Asp203Leu; Asp203Met; Asp203Asn; Asp203Pro; Asp203Gln; Asp203Arg; Asp203Ser; Asp203Thr; Asp203Val; Asp203Trp; Asp203Tyr; Lys204Ala; Lys204Cys; Lys204Asp; Lys204Glu; Lys204Phe; Lys204Gly; Lys204His; Lys204lle; Lys204Leu; Lys204Met; Lys204Asn; Lys204Pro; Lys204Gln; Lys204Arg; Lys204Ser; Lys204Thr; Lys204Val; Lys204Trp; Lys204Tyr; Thr205Ala; Thr205Cys; Thr205Asp; Thr205Glu; Thr205Phe; Thr205Gly; Thr205His; Thr205lle; Thr205Lys; Thr205Leu; Thr205Met; Thr205Asn; Thr205Pro; Thr205Gln; Thr205Arg; Thr205Ser; Thr205Val; Thr205Trp; Thr205Tyr; Gly206Ala; Gly206Cys; Gly206Asp; Gly206Glu; Gly206Phe; Gly206His; Gly206lle; Gly206Lys; Gly206Leu; Gly206Met; Gly206Asn; Gly206Pro; Gly206Gln; Gly206Arg; Gly206Ser; Gly206Thr; Gly206Val; Gly206Trp; Gly206Tyr; Ala207Cys; Ala207Asp; Ala207Glu; Ala207Phe; Ala207Gly; Ala207His; Ala207lle; Ala207Lys; Ala207Leu; Ala207Met; Ala207Asn; Ala207Pro; Ala207Gln; Ala207Arg; Ala207Ser; Ala207Thr; Ala207Val; Ala207Trp; Ala207Tyr; Ala208Cys; Ala208Asp; Ala208Glu; Ala208Phe; Ala208Gly; Ala208His; Ala208lle; Ala208Lys; Ala208Leu; Ala208Met; Ala208Asn; Ala208Pro; Ala208Gln; Ala208Arg; Ala208Ser; Ala208Thr; Ala208Val; Ala208Trp; Ala208Tyr; Ser209Ala; Ser209Cys; Ser209Asp; Ser209Glu; Ser209Phe; Ser209Gly; Ser209His; Ser209lle; Ser209Lys; Ser209Leu; Ser209Met; Ser209Asn; Ser209Pro; Ser209Gln; Ser209Arg; Ser209Thr; Ser209Val; Ser209Trp; Ser209Tyr; Tyr210Ala; Tyr210Cys; Tyr210Asp; Tyr210Glu; Tyr210Phe; Tyr210Gly; Tyr210His; Tyr210lle; Tyr210Lys; Tyr210Leu; Tyr210Met; Tyr210Asn; Tyr210Pro; Tyr210Gln; Tyr210Arg; Tyr210Ser; Tyr210Thr; Tyr210Val; Tyr210Trp; Gly21 1Ala; Gly21 1 Cys; Gly21 1Asp; Gly21 1 Glu; Gly21 1 Phe; Gly21 1 His; Gly21 1 lie; Gly21 1 Lys; Gly21 1 Leu; Gly21 1 Met; Gly21 1Asn; Gly21 1 Pro; Gly21 1 Gln; Gly21 1Arg; Gly211 Ser; Gly21 1Thr; Gly21 1Val; Gly21 1 Trp; Gly21 1 Tyr; Thr212Ala; Thr212Cys; Thr212Asp; Thr212Glu; Thr212Phe; Thr212Gly; Thr212His; Thr212lle; Thr212Lys; Thr212Leu; Thr212Met; Thr212Asn; Thr212Pro; Thr212Gln; Thr212Arg; Thr212Ser; Thr212Val; Thr212Trp; Thr212Tyr; Arg213Ala; Arg213Cys; Arg213Asp; Arg213Glu; Arg213Phe; Arg213Gly; Arg213His; Arg213lle; Arg213Lys; Arg213Leu; Arg213Met; Arg213Asn; Arg213Pro; Arg213Gln; Arg213Ser; Arg213Thr; Arg213Val; Arg213Trp; Arg213Tyr; Asn214Ala; Asn214Cys; Asn214Asp; Asn214Glu; Asn214Phe; Asn214Gly; Asn214His; Asn214lle; Asn214Lys; Asn214Leu; Asn214Met; Asn214Pro; Asn214Gln; Asn214Arg; Asn214Ser; Asn214Thr; Asn214Val; Asn214Trp; Asn214Tyr; Asp215Ala; Asp215Cys; Asp215Glu; Asp215Phe; Asp215Gly; Asp215His; Asp215lle; Asp215Lys; Asp215Leu; Asp215Met; Asp215Asn; Asp215Pro; Asp215Gln; Asp215Arg; Asp215Ser; Asp215Thr; Asp215Val; Asp215Trp; Asp215Tyr; He216Ala; He216Cys; He216Asp; He216Glu; He216Phe; He216Gly; He216His; He216Lys; He216Leu; He216Met; He216Asn; He216Pro; He216Gln; He216Arg; He216Ser; He216Thr; He216Val; He216Trp; He216Tyr; Ala217Cys; Ala217Asp; Ala217Glu; Ala217Phe; Ala217Gly; Ala217His; Ala217lle; Ala217Lys; Ala217Leu; Ala217Met; Ala217Asn; Ala217Pro; Ala217Gln; Ala217Arg; Ala217Ser; Ala217Thr; Ala217Val; Ala217Trp; Ala217Tyr; He218Ala; He218Cys; He218Asp; He218Glu; He218Phe; He218Gly; He218His; He218Lys; He218Leu; He218Met; He218Asn; He218Pro; He218Gln; He218Arg; He218Ser; He218Thr; He218Val; He218Trp; He218Tyr; He219Ala; He219Cys; He219Asp; He219Glu; He219Phe; He219Gly; He219His; He219Lys; He219Leu; He219Met; He219Asn; He219Pro; He219Gln; He219Arg; He219Ser; He219Thr; He219Val; He219Trp; He219Tyr; Trp220Ala; Trp220Cys; Trp220Asp; Trp220Glu; Trp220Phe; Trp220Gly; Trp220His; Trp220lle; Trp220Lys; Trp220Leu; Trp220Met; Trp220Asn; Trp220Pro; Trp220Gln; Trp220Arg; Trp220Ser; Trp220Thr; Trp220Val; Trp220Tyr; Pro221Ala; Pro221 Cys; Pro221Asp; Pro221 Glu; Pro221 Phe; Pro221 Gly; Pro221 His; Pro221 lle; Pro221 Lys; Pro221 Leu; Pro221 Met; Pro221Asn; Pro221 Gln; Pro221Arg; Pro221 Ser; Pro221 Thr; Pro221Val; Pro221 Trp; Pro221Tyr; Pro222Ala; Pro222Cys; Pro222Asp; Pro222Glu; Pro222Phe; Pro222Gly; Pro222His; Pro222lle; Pro222Lys; Pro222Leu; Pro222Met; Pro222Asn; Pro222Gln; Pro222Arg; Pro222Ser; Pro222Thr; Pro222Val; Pro222Trp; Pro222Tyr; Lys223Ala; Lys223Cys; Lys223Asp; Lys223Glu; Lys223Phe; Lys223Gly; Lys223His; Lys223lle; Lys223Leu; Lys223Met; Lys223Asn; Lys223Pro; Lys223Gln; Lys223Arg; Lys223Ser; Lys223Thr; Lys223Val; Lys223Trp; Lys223Tyr; Gly224Ala; Gly224Cys; Gly224Asp; Gly224Glu; Gly224Phe; Gly224His; Gly224lle; Gly224Lys; Gly224Leu; Gly224Met; Gly224Asn; Gly224Pro; Gly224Gln; Gly224Arg; Gly224Ser; Gly224Thr; Gly224Val; Gly224Trp; Gly224Tyr; Asp225Ala; Asp225Cys; Asp225Glu; Asp225Phe; Asp225Gly; Asp225His; Asp225lle; Asp225Lys; Asp225Leu; Asp225Met; Asp225Asn; Asp225Pro; Asp225Gln; Asp225Arg; Asp225Ser; Asp225Thr; Asp225Val; Asp225Trp; Asp225Tyr; Pro226Ala; Pro226Cys; Pro226Asp; Pro226Glu; Pro226Phe; Pro226Gly; Pro226His; Pro226lle; Pro226Lys; Pro226Leu; Pro226Met; Pro226Asn; Pro226Gln; Pro226Arg; Pro226Ser; Pro226Thr; Pro226Val; Pro226Trp; Pro226Tyr; Val227Ala; Val227Cys; Val227Asp; Val227Glu; Val227Phe; Val227Gly; Val227His; Val227lle; Val227Lys; Val227Leu; Val227Met; Val227Asn; Val227Pro; Val227Gln; Val227Arg; Val227Ser; Val227Thr; Val227Trp; Val227Tyr; Val228Ala; Val228Cys; Val228Asp; Val228Glu; Val228Phe; Val228Gly; Val228His; Val228lle; Val228Lys; Val228Leu; Val228Met; Val228Asn; Val228Pro; Val228Gln; Val228Arg; Val228Ser; Val228Thr; Val228Trp; Val228Tyr; Leu229Ala; Leu229Cys; Leu229Asp; Leu229Glu; Leu229Phe; Leu229Gly; Leu229His; Leu229lle; Leu229Lys; Leu229Met; Leu229Asn; Leu229Pro; Leu229Gln; Leu229Arg; Leu229Ser; Leu229Thr; Leu229Val; Leu229Trp; Leu229Tyr; Ala230Cys; Ala230Asp; Ala230Glu; Ala230Phe; Ala230Gly; Ala230His; Ala230lle; Ala230Lys; Ala230Leu; Ala230Met; Ala230Asn; Ala230Pro; Ala230Gln; Ala230Arg; Ala230Ser; Ala230Thr; Ala230Val; Ala230Trp; Ala230Tyr; Val231Ala; Val231 Cys; Val231Asp; Val231 Glu; Val231 Phe; Val231 Gly; Val231 His; Val231 lle; Val231 Lys; Val231 Leu; Val231 Met; Val231Asn; Val231 Pro; Val231 Gln; Val231Arg; Val231 Ser; Val231Thr; Val231Trp; Val231Tyr; Leu232Ala; Leu232Cys; Leu232Asp; Leu232Glu; Leu232Phe; Leu232Gly; Leu232His; Leu232lle; Leu232Lys; Leu232Met; Leu232Asn; Leu232Pro; Leu232Gln; Leu232Arg; Leu232Ser; Leu232Thr; Leu232Val; Leu232Trp; Leu232Tyr; Ser233Ala; Ser233Cys; Ser233Asp; Ser233Glu; Ser233Phe; Ser233Gly; Ser233His; Ser233lle; Ser233Lys; Ser233Leu; Ser233Met; Ser233Asn; Ser233Pro; Ser233Gln; Ser233Arg; Ser233Thr; Ser233Val; Ser233Trp; Ser233Tyr; Ser234Ala; Ser234Cys; Ser234Asp; Ser234Glu; Ser234Phe; Ser234Gly; Ser234His; Ser234lle; Ser234Lys; Ser234Leu; Ser234Met; Ser234Asn; Ser234Pro; Ser234Gln; Ser234Arg; Ser234Thr; Ser234Val; Ser234Trp; Ser234Tyr; Arg235Ala; Arg235Cys; Arg235Asp; Arg235Glu; Arg235Phe; Arg235Gly; Arg235His; Arg235lle; Arg235Lys; Arg235Leu; Arg235Met; Arg235Asn; Arg235Pro; Arg235Gln; Arg235Ser; Arg235Thr; Arg235Val; Arg235Trp; Arg235Tyr; Asp236Ala; Asp236Cys; Asp236Glu; Asp236Phe; Asp236Gly; Asp236His; Asp236lle; Asp236Lys; Asp236Leu; Asp236Met; Asp236Asn; Asp236Pro; Asp236Gln; Asp236Arg; Asp236Ser; Asp236Thr; Asp236Val; Asp236Trp; Asp236Tyr; Lys237Ala; Lys237Cys; Lys237Asp; Lys237Glu; Lys237Phe; Lys237Gly; Lys237His; Lys237lle; Lys237Leu; Lys237Met; Lys237Asn; Lys237Pro; Lys237Gln; Lys237Arg; Lys237Ser; Lys237Thr; Lys237Val; Lys237Trp; Lys237Tyr; Lys238Ala; Lys238Cys; Lys238Asp; Lys238Glu; Lys238Phe; Lys238Gly; Lys238His; Lys238lle; Lys238Leu; Lys238Met; Lys238Asn; Lys238Pro; Lys238Gln; Lys238Arg; Lys238Ser; Lys238Thr; Lys238Val; Lys238Trp; Lys238Tyr; Asp239Ala; Asp239Cys; Asp239Glu; Asp239Phe; Asp239Gly; Asp239His; Asp239lle; Asp239Lys; Asp239Leu; Asp239Met; Asp239Asn; Asp239Pro; Asp239Gln; Asp239Arg; Asp239Ser; Asp239Thr; Asp239Val; Asp239Trp; Asp239Tyr; Ala240Cys; Ala240Asp; Ala240Glu; Ala240Phe; Ala240Gly; Ala240His; Ala240lle; Ala240Lys; Ala240Leu; Ala240Met; Ala240Asn; Ala240Pro; Ala240Gln; Ala240Arg; Ala240Ser; Ala240Thr; Ala240Val; Ala240Trp; Ala240Tyr; Lys241Ala; Lys241 Cys; Lys241Asp; Lys241Glu; Lys241 Phe; Lys241 Gly; Lys241 His; Lys241 lle; Lys241 Leu; Lys241 Met; Lys241Asn; Lys241 Pro; Lys241Gln; Lys241Arg; Lys241 Ser; Lys241Thr; Lys241Val; Lys241Trp; Lys241Tyr; Tyr242Ala; Tyr242Cys; Tyr242Asp; Tyr242Glu; Tyr242Phe; Tyr242Gly; Tyr242His; Tyr242lle; Tyr242Lys; Tyr242Leu; Tyr242Met; Tyr242Asn; Tyr242Pro; Tyr242Gln; Tyr242Arg; Tyr242Ser; Tyr242Thr; Tyr242Val; Tyr242Trp; Asp243Ala; Asp243Cys; Asp243Glu; Asp243Phe; Asp243Gly; Asp243His; Asp243lle; Asp243Lys; Asp243Leu; Asp243Met; Asp243Asn; Asp243Pro; Asp243Gln; Asp243Arg; Asp243Ser; Asp243Thr; Asp243Val; Asp243Trp; Asp243Tyr; Asp244Ala; Asp244Cys; Asp244Glu; Asp244Phe; Asp244Gly; Asp244His; Asp244lle; Asp244Lys; Asp244Leu; Asp244Met; Asp244Asn; Asp244Pro; Asp244Gln; Asp244Arg; Asp244Ser; Asp244Thr; Asp244Val; Asp244Trp; Asp244Tyr; Lys245Ala; Lys245Cys; Lys245Asp; Lys245Glu; Lys245Phe; Lys245Gly; Lys245His; Lys245lle; Lys245Leu; Lys245Met; Lys245Asn; Lys245Pro; Lys245Gln; Lys245Arg; Lys245Ser; Lys245Thr; Lys245Val; Lys245Trp; Lys245Tyr; Leu246Ala; Leu246Cys; Leu246Asp; Leu246Glu; Leu246Phe; Leu246Gly; Leu246His; Leu246lle; Leu246Lys; Leu246Met; Leu246Asn; Leu246Pro; Leu246Gln; Leu246Arg; Leu246Ser; Leu246Thr; Leu246Val; Leu246Trp; Leu246Tyr; He247Ala; He247Cys; He247Asp; He247Glu; He247Phe; He247Gly; He247His; He247Lys; He247Leu; He247Met; He247Asn; He247Pro; He247Gln; He247Arg; He247Ser; He247Thr; He247Val; He247Trp; He247Tyr; Ala248Cys; Ala248Asp; Ala248Glu; Ala248Phe; Ala248Gly; Ala248His; Ala248lle; Ala248Lys; Ala248Leu; Ala248Met; Ala248Asn; Ala248Pro; Ala248Gln; Ala248Arg; Ala248Ser; Ala248Thr; Ala248Val; Ala248Trp; Ala248Tyr; Glu249Ala; Glu249Cys; Glu249Asp; Glu249Phe; Glu249Gly; Glu249His; Glu249lle; Glu249Lys; Glu249Leu; Glu249Met; Glu249Asn; Glu249Pro; Glu249Gln; Glu249Arg; Glu249Ser; Glu249Thr; Glu249Val; Glu249Trp; Glu249Tyr; Ala250Cys; Ala250Asp; Ala250Glu; Ala250Phe; Ala250Gly; Ala250His; Ala250lle; Ala250Lys; Ala250Leu; Ala250Met; Ala250Asn; Ala250Pro; Ala250Gln; Ala250Arg; Ala250Ser; Ala250Thr; Ala250Val; Ala250Trp; Ala250Tyr; Thr251Ala; Thr251 Cys; Thr251Asp; Thr251 Glu; Thr251 Phe; Thr251 Gly; Thr251 His; Thr251 lle; Thr251 Lys; Thr251 Leu; Thr251 Met; Thr251Asn; Thr251 Pro; Thr251 Gln; Thr251Arg; Thr251 Ser; Thr251Val; Thr251Trp; Thr251Tyr; Lys252Ala; Lys252Cys; Lys252Asp; Lys252Glu; Lys252Phe; Lys252Gly; Lys252His; Lys252lle; Lys252Leu; Lys252Met; Lys252Asn; Lys252Pro; Lys252Gln; Lys252Arg; Lys252Ser; Lys252Thr; Lys252Val; Lys252Trp; Lys252Tyr; Val253Ala; Val253Cys; Val253Asp; Val253Glu; Val253Phe; Val253Gly; Val253His; Val253lle; Val253Lys; Val253Leu; Val253Met; Val253Asn; Val253Pro; Val253Gln; Val253Arg; Val253Ser; Val253Thr; Val253Trp; Val253Tyr; Val254Ala; Val254Cys; Val254Asp; Val254Glu; Val254Phe; Val254Gly; Val254His; Val254lle; Val254Lys; Val254Leu; Val254Met; Val254Asn; Val254Pro; Val254Gln; Val254Arg; Val254Ser; Val254Thr; Val254Trp; Val254Tyr; Met255Ala; Met255Cys; Met255Asp; Met255Glu; Met255Phe; Met255Gly; Met255His; Met255lle; Met255Lys; Met255Leu; Met255Asn; Met255Pro; Met255Gln; Met255Arg; Met255Ser; Met255Thr; Met255Val; Met255Trp; Met255Tyr; Lys256Ala; Lys256Cys; Lys256Asp; Lys256Glu; Lys256Phe; Lys256Gly; Lys256His; Lys256lle; Lys256Leu; Lys256Met; Lys256Asn; Lys256Pro; Lys256Gln; Lys256Arg; Lys256Ser; Lys256Thr; Lys256Val; Lys256Trp; Lys256Tyr; Ala257Cys; Ala257Asp; Ala257Glu; Ala257Phe; Ala257Gly; Ala257His; Ala257lle; Ala257Lys; Ala257Leu; Ala257Met; Ala257Asn; Ala257Pro; Ala257Gln; Ala257Arg; Ala257Ser; Ala257Thr; Ala257Val; Ala257Trp; Ala257Tyr; Leu258Ala; Leu258Cys; Leu258Asp; Leu258Glu; Leu258Phe; Leu258Gly; Leu258His; Leu258lle; Leu258Lys; Leu258Met; Leu258Asn; Leu258Pro; Leu258Gln; Leu258Arg; Leu258Ser; Leu258Thr; Leu258Val; Leu258Trp; Leu258Tyr; Asn259Ala; Asn259Cys; Asn259Asp; Asn259Glu; Asn259Phe; Asn259Gly; Asn259His; Asn259lle; Asn259Lys; Asn259Leu; Asn259Met; Asn259Pro; Asn259Gln; Asn259Arg; Asn259Ser; Asn259Thr; Asn259Val; Asn259Trp; Asn259Tyr; Met260Ala; Met260Cys; Met260Asp; Met260Glu; Met260Phe; Met260Gly; Met260His; Met260lle; Met260Lys; Met260Leu; Met260Asn; Met260Pro; Met260Gln; Met260Arg; Met260Ser; Met260Thr; Met260Val; Met260Trp; Met260Tyr; Asn261Ala; Asn261 Cys; Asn261Asp; Asn261 Glu; Asn261 Phe; Asn261 Gly; Asn261 His; Asn261 lle; Asn261 Lys; Asn261Leu; Asn261 Met; Asn261 Pro; Asn261 Gln; Asn261Arg; Asn261Ser; Asn261Thr; Asn261Val; Asn261Trp; Asn261Tyr; Gly262Ala; Gly262Cys; Gly262Asp; Gly262Glu; Gly262Phe; Gly262His; Gly262lle; Gly262Lys; Gly262Leu; Gly262Met; Gly262Asn; Gly262Pro; Gly262Gln; Gly262Arg; Gly262Ser; Gly262Thr; Gly262Val; Gly262Trp; Gly262Tyr; Lys263Ala; Lys263Cys; Lys263Asp; Lys263Glu; Lys263Phe; Lys263Gly; Lys263His; Lys263lle; Lys263Leu; Lys263Met; Lys263Asn; Lys263Pro; Lys263Gln; Lys263Arg; Lys263Ser; Lys263Thr; Lys263Val; Lys263Trp; Lys263Tyr; Met 264Ala; Met 264Cys; Met 264Asp; Met 264Glu; Met 264Phe; Met 264Gly; Met 264His; Met 264lle; Met 264Lys; Met 264Leu; Met 264Asn; Met 264Pro; Met 264Gln; Met 264Arg; Met 264Ser; Met 264Thr; Met 264Val; Met 264Trp; Met 264Tyr; Asn 265Ala; Asn 265Cys; Asn 265Asp; Asn 265Glu; Asn 265Phe; Asn 265Gly; Asn 265His; Asn 265lle; Asn 265Lys; Asn 265Leu; Asn 265Met; Asn 265Pro; Asn 265Gln; Asn 265Arg; Asn 265Ser; Asn 265Thr; Asn 265Val; Asn 265Trp; Asn 265Tyr; Gly 266Ala; Gly 266Cys; Gly 266Asp; Gly 266Glu; Gly 266Phe; Gly 266His; Gly 266lle; Gly 266Lys; Gly 266Leu; Gly 266Met; Gly 266Asn; Gly 266Pro; Gly 266Gln; Gly 266Arg; Gly 266Ser; Gly 266Thr; Gly 266Val; Gly 266Trp; Gly 266Tyr; Lys267Ala; Lys267Cys; Lys267Asp; Lys267Glu; Lys267Phe; Lys267Gly; Lys267His; Lys267lle; Lys267Leu; Lys267Met; Lys267Asn; Lys267Pro; Lys267Gln; Lys267Arg; Lys267Ser; Lys267Thr; Lys267Val; Lys267Trp; and Lys267Tyr. In some embodiments, SEQ ID NO: 1 may have a Met and/or Thr preceeding the first residue of the sequence. These residues may be similarly mutated as above.

In all of these mutants, the numbering of residues corresponds to SEQ ID NO: 1. These residue numbers may be converted to Ambler numbers (Ambler et al., 1991 , A standard numbering scheme for the Class A β-lactamases, Biochem. J. 276:269-272, the contents of which are hereby incorporated by reference) through use of any conventional bioinformatic method, for example by using BLAST (Basic Local Alignment Search Tools) or FASTA (FAST-AM). For example, residue 244 corresponds to Ambler 276. For example, the following conversions may be used:

Ambler Classification No. SEQ ID NO: 1 Residue

R244 R213

S266 S234

D276 D244

Furthermore, percent identity may also be assessed with these conventional bioinformatic methods.

In one aspect, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more of the following mutations of Ambler classification: F33X, Q135X, G156X, A232X, A237X, A238X, S240X, T243X, R244X, S266X, and D276X, wherein X is any naturally-occurring amino acid. In some embodiments, X is a naturally occurring hydrophilic or hydrophobic amino acid residue or a non- classical amino acid.

In another aspect, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more of the following mutations of Ambler classification: a hydrophobic residue other than phenylalanine (F) at position 33; a hydrophobic residue other than glutamine (Q) at position 135; a hydrophilic residue other than glycine (G) at position 156; a hydrophobic residue other than alanine (A) at position 232; a hydrophilic residue other than alanine (A) at position 237; a hydrophobic or hydrophilic residue other than alanine (A) at position 238; a hydrophilic residue other than serine (S) at position 240; a hydrophobic residue other than threonine (T) at position 243; a hydrophobic residue other than arginine (R) at position 244; a hydrophilic residue other than serine (S) at position 266; and a hydrophilic residue other than aspartate (D) at position 276.

As used throughout, a hydrophilic amino acid residue may include a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or an aromatic, polar and positively charged hydrophilic including histidine (H). As used throughout, a hydrophobic amino acid residue may include a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

Mutations may be made to the gene sequence of a beta-lactamase (e.g. SEQ ID NOs: 2 and 4) by reference to the genetic code, including taking into account codon degeneracy.

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises one or more of the following mutations at positions of Ambler classification: F33Y, Q135M, G156R, A232G, A237S, A238G or T, S240P or D, T243I, R244T, S266N, D276N or R or K. In one embodiment, the beta-lactamases and/or pharmaceutical compositions comprise Q135M. In another embodiment, the beta-lactamases and/or pharmaceutical compositions comprise G156R and A238T. In another embodiment, the beta-lactamases and/or pharmaceutical compositions comprise F33Y and D276N. In still another embodiment, the beta-lactamases and/or pharmaceutical compositions comprise F33Y, S240P, and D276N. In one embodiment, the beta- lactamases and/or pharmaceutical compositions comprise F33Y, A238T, and D276N. In another embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, and S240D. In a further embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D, and R244T. In another embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D, and D276R. In one embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D, and D276K. In one embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D, and Q135M. In one embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A238T. In one embodiment, the beta- lactamases and/or pharmaceutical compositions comprise T243I, S266N, and D276N. In one embodiment, the beta-lactamases and/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D, and D276N.

In various embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises one or more of the following mutations:

G156R A238T IS235

F33Y D276N IS158

F33Y S240P D276N IS230 (or IS181)

F33Y A238T D276N IS232 (or IS180)

I72S Q135M T160F (Block 1 mutants) IS227

A232G A237S A238G S240D (Block 2 mutants) IS191

A232G A237S A238G S240D R244T IS229

A232G A237S A238G S240D D276R IS219

A232G A237S A238G S240D D276K IS221

A232G A237S A238G S240D Q135M IS224

A238T IS233

T243I S266N D276N IS234 (or IS176)

A232G A237S A238G S240D D276N IS288 (or P4A)

In various embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 60% sequence identity with one or more of the mutants provided in the table directly above.

In illustrative embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and the following of Ambler classification: a residue other than aspartate (D) at position 276.

In illustrative embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 90%, or 95%, or 97%, or 99% sequence identity with SEQ ID NO: 1 and a hydrophilic amino acid residue other than aspartic acid (D) at a position corresponding to position 276 according to Ambler classification, wherein: the hydrophilic amino acid residue is asparagine (N) and the beta-lactamase hydrolyzes ceftriaxone substantially more efficiently than a beta-lactamase of SEQ ID NO: 1 that has an aspartic acid (D) at a position corresponding to position 276 according to Ambler classification.

In illustrative embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 90%, or 95%, or 97%, or 99% sequence identity with SEQ ID NO: 1 and a hydrophilic amino acid residue other than aspartic acid (D) at a position corresponding to position 276 according to Ambler classification, wherein: the hydrophilic amino acid residue is arginine (R) and the beta-lactamase hydrolyzes ceftriaxone substantially more efficiently than a beta-lactamase of SEQ ID NO: 1 that has an aspartic acid (D) at a position corresponding to position 276 according to Ambler classification.

In some embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 90%, or 95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 5, i.e. P3A:

SEQ ID NO: 5

TEMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQ

RITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDE

VTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALI

RAGVPDGWEVADKTGAASYGTRNDIAIIWPPKGDPWLAVLSSRDKKDAKYDNKLIAEATKW

MKALNMNGK.

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 5.

An illustrative polynucleotide of the invention is SEQ ID NO: 6, which is the full nucleotide sequence of P3A:

SEQ ID NO: 6:

atgactgagatgaaagatgattttgcgaagctggaagaacagtttgacgcaaaattgggcattttcgcgttggacacgg

gtacgaatcgtacggttgcctaccgtccggacgagcgcttcgccttcgcgagcacgatcaaagccctgaccgtcggcg

tgctgctccagcaaaagagcatcgaggacctgaaccagcgcattacctacacccgtgatgatctggtgaactataatc

cgatcaccgagaaacacgttgataccggtatgaccctgaaagaactggcagatgcaagcctgcgctacagcgataa

cgcggctcagaatctgattctgaagcaaatcggtggtccggagagcttgaagaaagaactgcgtaaaatcggcgatg

aagtcactaatccggagcgttttgagccggagctgaacgaagtgaatccgggtgaaacgcaagacacgagcaccg

cgcgtgcgcttgtcacctccctgcgcgctttcgcactggaagataagctgccgtcggagaaacgcgagctgctgatcg

actggatgaagcgcaatacgaccggcgacgcgctgattcgtgcgggcgttccggacggttgggaagtggctgacaa

gaccggtgcggcgagctacggcacccgtaacgatatcgcgatcatttggccacctaaaggtgacccggtcgtgctgg

ccgtactgagcagccgtgacaagaaagacgcaaagtatgataacaagctgattgcagaggcgaccaaagttgttat

gaaggcactgaacatgaatggtaag

In some embodiments, the polynucleotide of the present invention has at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 6.

In illustrative embodiments, the beta-lactamases and/or pharmaceutical compositions comprise an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and the following of Ambler classification: a hydrophobic residue other than alanine (A) at position 232; a hydrophilic residue other than alanine (A) at position 237; a hydrophobic residue other than alanine (A) at position 238; a hydrophilic residue other than serine (S) at position 240; and a hydrophilic residue other than aspartate (D) at position 276. In some embodiments, the hydrophobic residue other than alanine (A) at position 232 is glycine (G). In some embodiments, the hydrophilic residue other than alanine (A) at position 237 is serine (S). In some embodiments, the hydrophobic residue other than alanine (A) at position 238 is glycine (G). In some embodiments, the hydrophilic residue other than serine (S) at position 240 is aspartate (D). In some embodiments, the other than aspartate (D) at position 276 is asparagine (N). In some embodiments, the beta-lactamase and/or pharmaceutical composition comprises one or more of A232G, A237S, A238G, S240D, and D276N. In some embodiments, the beta-lactamase and/or pharmaceutical composition comprises all of A232G, A237S, A238G, S240D, and D276N, the sequence of which is SEQ ID NO: 7, i.e. P4A. In some embodiments, the beta- lactamase and/or pharmaceutical composition comprises an amino acid sequence having at least 90%, or 95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 7.

SEQ ID NO: 7

EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVL

LQQKSIEDLNQRITTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQ

NLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALV

TSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGS

GDYGTRNDIAIIWPPKGDPWLAVLSSRDKKDAKYDNKLIAEATKWMKALN

MNGK

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 7. SEQ ID NO: 8, is derived from SEQ ID NO: 7, and further includes the signal and the addition of the QASKT amino acids (the coding region is underlined):

MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAQASKTEMKDDFAKLEEQFDAKLG

IFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLV

NYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGD

EVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDW

MKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPWLAVL

SSRDKKDAKYDNKLIAEATKWMKALNMNGK

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 8.

In some embodiments, the beta-lactamase and/or pharmaceutical composition comprises an amino acid sequence having at least 90%, or 95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 8.

An illustrative polynucleotide of the invention is SEQ ID NO: 9, which is the full nucleotide sequence of A232G, A237S, A238G, S240D, and D276N mutant, Hind III site (AAGCTT-in bold) and additional K and T amino acids. In some embodiments, the underlined portion of SEQ ID NO: 9, is omitted. The leader and additional nucleotides (Hind III site and K and T amino acids— for the addition of the amino acid sequence QASKT) are underlined.

atqattcaaaaacqaaaqcqqacaqtttcqttcaqacttqtqcttatqtqcacqctqttatttqtcaqtttqccq attacaaaaacatcaqcqcaaqcttccaaqacqqaqatqaaaqatqattttqcaaaacttqaqqaaca atttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatg agcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatc tgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttga tacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctc attcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaa atcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagca agagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagctttta atcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaa gtgggtgataaaactggaagcggagattatggaacccggaatgacattgccatcatttggccgccaaaa ggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgataataaacttatt gcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the polynucleotide of the present invention has at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 9 (with or without the underlined portion).

In various aspects, the beta-lactamases polypeptide has the sequence of SEQ ID NO: 10 (i.e., P2A) or is derived by one or more mutations of SEQ ID NO: 10:

ETGTISISQLNKNVWVHTELGYFNGEAVPSNGLVLNTSKGLVLVDSSWDNK

LTKELIEMVEKKFQKRVTDVIITHAHADRIGGITALKERGIKAHSTALTAELAK

NSGYEEPLGDLQTITSLKFGNTKVETFYPGKGHTEDNIWWLPQYQILAGG

CLVKSAEAKDLGNVADAYVNEWSTSIENVLKRYGNINSWPGHGEVGDKG

LLLHTLDLLK.

In some embodiments, the beta-lactamase polypeptide produced by methods of the invention comprises an amino acid sequence having at least about 60% (e.g. about 60%, or about 61 %, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71 %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81 %, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91 %, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with SEQ ID NO: 10.

In some embodiments, the beta-lactamase and/or pharmaceutical composition comprises an amino acid sequence having at least 90%, or 95%, or 97%, or 99%, or 100% sequence identity with SEQ ID NO: 10.

Additional sequences of beta-lactamases including P1A, P2A, P3A, and P4A and derivatives thereof are described for example, in WO 2011/148041 and PCT/US2015/026457, the entire contents of which are hereby incorporated by reference.

The invention provides for polynucleotides encoding a beta-lactamase polypeptide, including, for example, vectors, comprising such polynucleotides. Such polynucleotides may further comprise, in addition to sequences encoding the beta-lactamases of the invention, one or more expression control elements. For example, the polynucleotide, may comprise one or more promoters or transcriptional enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, as expression control elements. In an embodiment, the polynucleotide includes expression control elements that direct expression of the beta- lactamase in the cytoplasm.

The polynucleotide may be inserted within a suitable vector, which is utilized to transform a suitable host cell such as an E. coli cell for expression. The vector may be any self-replicating DNA molecule that can transfer a DNA between host cells, including, for example, a plasmid cloning vector. In some embodiments, the vector can remain episomal or become chromosomally integrated, as long as the insert encoding the therapeutic agent can be transcribed. Vectors can be constructed by standard recombinant DNA technology. Vectors can be, for example, plasmids, phages, cosmids, phagemids, viruses, or any other types known in the art, which are used for replication and expression in prokaryotic or eukaryotic cells (e.g. an adenovirus; a retrovirus; a lentivirus; an scAAV; pGEX vector; pET vector; and pHT vector). Exemplary vectors that may be used include, for example, the pAVEO11 vector. Preparations of the pAVEO11 vector is described in EP Patent No. 0502637, EP Patent No. 2386642, and US Patent No. 6,537,779, the entire contents of which are hereby incorporated by reference. It will be appreciated by one of skill in the art that a wide variety of components known in the art (such as expression control elements) may be included in such vectors, including a wide variety of transcription signals, such as promoters and other sequences that regulate the binding of RNA polymerase onto the promoter. Any promoter known to be effective in £ co// cells in which the vector will be expressed can be used to initiate expression of the therapeutic agent. In one embodiment, the promoter is effective for directing expression of the beta-lactamase polypeptide in the cytoplasm. Suitable promoters may be inducible or constitutive. Examples of suitable promoters include, for example, the pET system (INVITROGEN), lac promoter, tac, trc, T7, T7A3 promoter, PhoA, Phage lambda pR, lambda pL promoter (see, e.g. J Ind Microbiol Biotechnoi (2012) 39:383-399; Curr Opin Biotech 2001 , 12: 195, the contents of which are hereby incorporated by reference), Pspac, PgroES, Pgsi, Plux and amyQ promoter and/or amyQ signal peptide from Bacillus amyloliquefaciens (by way of non-limiting example Gen Bank ID No. J01542.1 , the contents of which are hereby incorporated by reference). The promoter may be inducible (e.g. via IPTG, metabolites, temperature). In one embodiment, the cytoplasmic expression of the beta-lactamase polypeptide is driven by the IPTG inducible Lacl promoter. In one embodiment, cytoplasmic expression of the beta-lactamase polypeptide is induced by adding IPTG to the bacterial culture.

In various embodiments, the transformed E. coli cell is grown for a time under conditions sufficient to produce cytoplasmic expression of the beta-lactamase polypeptide. Any type of media that will support growth and reproduction of £ coli cell in cultures is useful for practicing the method of the invention. After growth of the cultures, the £. coli cell is typically lysed using osmotic shock, sonication or other standard means, and the expressed beta-lactamase polypeptide is isolated from the soluble fraction. Any protein purification method may be employed for this purpose, such as dialysis, gel filtration, ion exchange chromatography, affinity chromatography, electrophoresis, or a combination of steps.

In various embodiments, the beta-lactamases produced by methods of the invention possess functional characteristics that make them desirable for a variety of uses, including therapeutic uses. Methods of characterizing beta-lactamases are known in the art (e.g. nitrocefin assay as described by O'Callaghan, ef a/. Antimicrob. Agents Chemother, 1 :283-288; the various methods of Viswanatha ef al. Methods Mol Med. 2008;142:239-60).

In one embodiment, the beta-lactamases produced by methods of the invention hydrolyze one or more of penicillins and cephalosporins. As used throughout, penicillins include, for example, Amoxicillin (e.g. NOVAMOX, AMOXIL); Ampicillin (e.g. PRINCIPEN); Azlocillin; Carbenicillin (e.g. GEOCILLIN); Cloxacillin (e.g. TEGOPEN); Dicloxacillin (e.g. DYNAPEN); Flucloxacillin (e.g. FLOXAPEN); Mezlocillin (e.g. MEZLIN); Methicillin (e.g. STAPHCILLIN); Nafcillin (e.g. UNIPEN); Oxacillin (e.g. PROSTAPHLIN); Penicillin G (e.g. PENTIDS or PFIZERPEN); Penicillin V (e.g. VEETIDS (PEN-VEE-K)); Piperacillin (e.g. PIPRACIL); Temocillin (e.g. NEGABAN); and Ticarcillin (e.g. TICAR). As used throughout, cephalosporins include, for example, a first generation cephalosporin (e.g. Cefadroxil (e.g. DURICEF); Cefazolin (e.g. ANCEF); Ceftolozane, Cefalotin/Cefalothin (e.g. KEFLIN); Cefalexin (e.g. KEFLEX); a second generation cephalosporin (e.g. Cefaclor (e.g. DISTACLOR); Cefamandole (e.g. MANDOL); Cefoxitin (e.g. MEFOXIN); Cefprozil (e.g. CEFZIL); Cefuroxime (e.g. CEFTIN, ZINNAT)); a third generation cephalosporin (e.g. Cefixime (e.g. SUPRAX); Cefdinir (e.g. OMNICEF, CEFDIEL); Cefditoren (e.g. SPECTRACEF); Cefoperazone (e.g. CEFOBID); Cefotaxime (e.g. CLAFORAN); Cefpodoxime (e.g. VANTIN); Ceftazidime (e.g. FORTAZ); Ceftibuten (e.g. CEDAX) Ceftizoxime (e.g. CEFIZOX); and Ceftriaxone (e.g. ROCEPHIN)); a fourth generation cephalosporin (e.g. Cefepime (e.g. MAXIPIME)); or a fifth generation cephalosporin (e.g. Ceftaroline fosamil (e.g. TEFLARO); Ceftobiprole (e.g. ZEFTERA)). In a specific embodiment, cephalosporins include, for example, cefoperazone, ceftriaxone or cefazolin. In a specific embodiment, the inventive beta-lactamases have improved catalytic efficiency against cephalosporins as compared to SEQ ID NO: 1.

In various embodiments, the beta-lactamases possess desirable enzyme kinetic characteristics. For example, in some embodiments, the beta-lactamases possess a low KM for at least one cephalosporin, including, for example, a K_M of less than about 500 μΜ, or about 100 μΜ, or about 10 μΜ, or about 1 μΜ, or about 0.1 μΜ (100 nM), or about 0.01 μΜ (10 nM), or about 1 nM. For example, in some embodiments, the beta-lactamases possess a low KM for at least one penicillin, including, for example, a KM of less than about 500 μΜ, or about 100 μΜ, or about 10 μΜ, or about 1 μΜ, or about 0.1 μΜ (100 nM), or about 0.01 μΜ (10 nM), or about 1 nM. In various embodiments, the inventive beta-lactamases possess a high V_max for at least one cephalosporin, including, for example, V_max which is greater than about 100 s-1 , or about 1000 s-1 , or about 10000 s-1 , or about 100000 s-1 , or about 1000000 s-1. In various embodiments, the inventive beta-lactamases possess a high V_max for at least one penicillin, including, for example, V_max which is greater than about 100 s-1 , or about 1000 s-1 , or about 10000 s-1 , or about 100000 s-1 , or about 1000000 s-1. In various embodiments, the inventive beta- lactamases possess catalytic efficiency is greater than about 10⁶ M^"1 s^-1 for at least one cephalosporin. In various embodiments, the inventive beta-lactamases possess catalytic efficiency is greater than about 10⁶ M^"1 s^-1 for at least one penicillin. In various embodiments, the inventive beta-lactamases possess the desirable enzyme kinetic characteristics for at least one of either or both of cephalosporins and penicillins.

In various embodiments, the inventive beta-lactamases are stable and/or active in the Gl tract, e.g. in one or more of the mouth, esophagus, stomach, duodenum, small intestine, duodenum, jejunum, ileum, large intestine, colon transversum, colon descendens, colon ascendens, colon sigmoidenum, cecum, and rectum. In a specific embodiment, the beta-lactamase is stable in the large intestine, optionally selected from one or more of colon transversum, colon descendens, colon ascendens, colon sigmoidenum and cecum. In a specific embodiment, the beta-lactamase is stable in the small intestine, optionally selected from one or more of duodenum, jejunum, and ileum. In some embodiments, the beta-lactamase is resistant to proteases in the Gl tract, including for example, the small intestine. In some embodiments, the beta-lactamase is substantially active at a pH of about 6.0 to about 7.5, e.g. about 6.0, or about 6.1 , or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1 , or about 7.2, or about 7.3, or about 7.4, or about 7.5 (including, for example, via formulation, as described herein). In various embodiments, the beta- lactamases of the present invention are resistant to one or more beta-lactamase inhibitors, optionally selected from avibactam, tazobactam, sulbactam, and clavulanic acid. In some embodiments, stable refers to an enzyme that has a long enough half-life and maintains enough activity for therapeutic effectiveness.

This invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1: Production of Beta-Lactamases in Bacillus Strains

P1A-protein was produced by Bacillus subtilis RS310 production strain in approximately 10,000 liter fed-batch fermentation. The Bacillus subtilis RS310 strain was asporogenic, tryptophan auxotrophic and secreted P1A- protein into the culture broth. Specifi callly, cell culturing of the P1A-protein comprised two inoculum (1 %) expansion stages in shake flasks (WCB vial -> 100 mL -> 2 x 1200 mL) followed by a seed fermentation stage (220 L, 2.5 %). The main fed-batch fermentation was conducted in approximately 10,000 L working volume. The main fermentation was started as batch fermentation with an initial volume of 9,000 L of growth medium. After about 9 hours when most of glucose in the growth medium was consumed, feeding with a feed solution (approx. 1500 - 2000 L) containing glucose and phosphate was started. In order to keep glucose at adequate levels (0.5 - 5 mg/mL) during the feeding phase, predefined feeding profile was used, which may be adjusted during the process based on glucose measurements. The P1A protein was constitutively produced and secreted extracellularly into the culture broth.

During fermentation the critical operational parameters were monitored and controlled including glucose concentration, pH (7 ± 0.2), dissolved oxygen level (10 - 20 %), temperature (37 ± 1°C) and foam level. Stirring rate was controlled starting with gentle mixing and increasing to a maximum of 138 -145 rpm. Air flow into the vessel was adjusted to 0.5-1 vvm. Progression of fermentation was monitored by P1A content (enzyme activity measurement) and cell density measurements (OD 600 nm). The main fermentation achieved a P1A titer of about 1-1.2 mg/mL (by HPLC) typically after 16-22 hours. The final cell density was typically approximately OD 50 (d.w. 16-17 g/L). After completion of cultivation, the content of fermenter was cooled down to 11 ± 3 °C.

After fermentation the cells were removed from P1A-protein containing broth by continuous centrifugation followed by microfiltration. P1A containing filtrate was concentrated by ultrafiltration and P1A concentrate was further diafiltered, conditioned and passed through a disposable anion exchange filter cartridge in flow-through mode after which the filtrate was further diafiltered to remove NaCI. This prepared the solution for the following two stage P1A-protein crystallisation including; crystallisation, crystal harvesting, washing and dissolution. Finally, after the second crystallisation step, P1A-protein crystals were suspended in water and dissolved and final concentration of P1A-protein solution was adjusted. The protein solution was filtered (0.2 urn) to reduce bioburden and finally dispensed into sterile plastic containers, frozen and stored at -70°C.

Example 2: Intracellular Gene Design for the Expression of P3A β-Lactamase

The purpose of this study was to improve β-lactamase expression. To do so, the pAVEway™ advance protein expression system was employed in £ coli. P3A was used throughout this study for testing β-lactamase expression. The gene sequence for directing the intracellular expression of P3A is SEQ ID NO: 6.

The P3A gene was cloned into the pAVEway™ intercellular (cytoplasmic) construct, pAVE011 , and the plasmid was verified with PCR and DNA sequencing. The designed P3A expression construct provided a relatively homogeneous N-terminus with the N-terminal methionine removed about 95% of the time.

Following construction of the intercellular expression plasmid, the construct was transformed in the following £ coli strains: CLD977 (W3110 E. coli host) and CLD990 (BL21 £ coli host). After construction of the β-lactamase intracellular expression strains, P3A was expressed and characterized as further described in Examples 2 and 3, respectively.

Additionally, the P3A gene was cloned into the pAVEway™ periplasmic construct, pAVE029 + gene 1 or gene 7 (gene 1 and gene 7 are different secretion leaders). Again, the plasmid was verified with PCR and DNA sequencing.

Following construction of the periplasmic expression plasmid, the construct was transformed in the following E. coli strains: CLD981 (gene 1 leader, W3110 E. coli host) and CLD982 (gene 7 leader, BL21 £. coli host). After construction of the periplasmic β-lactamase expression strains, P3A was expressed and characterized as further described in Examples 2 and 3, respectively.

Example 3: P3A β-lactamase Fermentation

Duplicate fermentations were performed using intracellular expression strains CLD977 and CLD990, and periplasmic strains CLD981 and CLD982. Specifically, the fermentation analysis was carried out in 3 stages: Shake flask (SF) seed stage, Fermenter stage, and SDS-PAGE analysis stage. To carry out the SF seed stage, RCB vials were inoculated into duplicate shake flasks with standard media and incubated at 37°C, 200 rpm for approximately 10 hours. Next, purity and OD₆oo of the samples was determined (summarized in Table 1). Finally, the E. coli material was transferred from SF to a fermentation vessel.

Table 1. Results from the shake flask seed stage for intracellular strains CLD977 and CLD990. SF1 and SF2 correspond to duplicate reactions for CLD977 and CLD990, respectively.

The fermenter stage was conducted using the standard pAVEway™ intracellular protocol. Specifically, cultures were induced using 0.5 mM IPTG when ODeoo = 50 ± 5. After induction, fermentation continued for an additional 12 hours before shutdown. Purity of the samples was confirmed at both pre-induction and shutdown.

For CLD977, the fermentation control parameter steps were: i) Oxygen supplementation at 7.33 hours; ii) End of batch phase at 9.46 hours when feed started; iii) Induction at 10.27 hours when OD₆oo = 50.1 ; iv) Fermentation continued for a further 12 hours before shutdown.

As shown in Fig. 1 (a multi-fermenter computer system (MFCS) plot of CLD977 fermentation), at approximately 20 hours, the airflow began to fail, which was suspected to be due to pressure in the vessel. Also, shown in Fig. 1 , p02 fell below 20% at approximately 21 hours and 1.5 hours prior to shut down.

For CLD990, the fermentation control parameter steps were: i) Oxygen supplementation at 10.95 hours; ii) End of batch phase at 12.27 hours when feed started; iii) Induction at 13.14 hours when OD600 = 50.1 ; iv)

Fermentation continued for a further 12 hours before shutdown. A MFCS plot of CLD990 fermentation is shown in Fig. 2.

A MFCS plot of exit gas analysis of oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) for CLD977 and CLD990 fermentation is shown in Fig. 3. Similar profiles were observed for both strains with the delay seen on the CLD990 strain due to an observed longer batch phase. Profile at the end of CLD977 fermentation, without wishing to be bound by theory, was probably related to a reduced airflow in the vessel (exit filter blocked).

Biomass profiles for both strains were similar up to 12 hours post induction although the CLD990 strain was delayed due to the extended batch phase (see Fig. 4). This delay, without wishing to be bound by theory, may have been due to the lower SF OD₆oo or a reduced initial growth rate.

Gram staining was also performed for CLD977 and CLD990 at the end of batch phase and after fermentation was complete (see Fig. 5). Results indicate that the culture was pure and homogenous at the end of the culturing.

Following fermentation, selected time course samples from pre-induction to the end of fermentation were analyzed using SDS-PAGE (see Fig. 6 - 8) after samples were lysed, spun down, and resuspended.

As evidenced by the SDS-PAGE results, protein product levels in both strains were in excess of 10 g/L (see Fig. 6 and 7): CLD977 SDS PAGE indicated 12.1 - 14.0 g/L whereas CLD990 SDS PAGE indicated 13.2 - 13.7 g/L. Additionally, the CLD977 and CLD990 total protein products (after sonication) were mostly soluble (see Fig. 8). Compared to previous systems used to express β-lactamase (that yielded about 0.5 to 1 g/L), the methods of the present invention utilizing intracellular expression of β-lactamase in E. coli strains proved to be far superior. Contrary to prior studies which show periplasmic β-lactamase expression, attempts to express β-lactamase in the periplasmic domain were unsuccessful and led to biologically inactive β-lactamase (see Example 3).

Example 4: β-lactamase Activity of Fermentation Samples by the CENTA Method

P3A β-lactamase activity of the previously described fermentation samples (see Example 2) was analyzed using the CENTA method, which is described below. Throughout this method, different standards were used and are referred to as: Reference material (32 mg/mL); Standard curve: Reference standard material diluted x1000 (standards used were 0.6 mg/l, 0.8 mg/l, 1.0 mg/l, 1.5 mg/l, 2.0 mg/l and 4.0 mg/l made up from the x1000 stock); Control standard: Reference standard diluted to 1 mg/l and ran as a control; 1 mM CENTA stock solution: 25 mg CENTA lactamase substrate dissolved in 50 ml of 50 mM Sodium dihydrogen phosphate (stored at -20°C); and CENTA working solution: 3.34 ml of CENTA stock solution dissolved in 25 ml of Sodium dihydrogen phosphate.

The CENTA method employs a chromogenic cephalosporin that is readily hydrolyzed by β-lactamases and allows for kinetic studies and detection of the enzymes in crude extracts and chromatographic fractions (Bebrone, C. ef a/., (2001) Antimicrobial Agents and Chemotherapy, 45 (6) 1868-1871). This method is also useful since CENTA can be prepared from the commercially available drug, cephalothin. For this study, β- lactamase sample activity was monitored using a FFDB plate reader in the presence of a CENTA working solution. First, β-lactamase samples were diluted to 1 mg/l (Bradford assays were used to determine the concentration). Then, 50 μί. of each sample was loaded onto the plate and incubated for 20 min. at 25°C. Finally, 200 \ii of the CENTA working solution was added to each sample and the sample was read as follows: Plate reader settings: Temperature of measurement = 25°C; Shaking = slow; Time of shaking = 2 seconds; Time of measurement = 60 seconds; Number of readings = Every 3 seconds; and Wavelength = 405 nm.

The hydrolysis of CENTA was monitored by continuously recording the absorbance variation at 405 nm (appearance of the expulsed chromophore). Results from this assay are presented in Figs. 9 - 19 and summarized in Tables 2 - 4.

The CENTA experiments were split into 3 assay plates. Assay plate 1 corresponded to: CLD981 12h, 24h, 48h, osmotic shock buffer 1 (OS1 ) 24h, and osmotic shock buffer 2 (OS2) 24h post induction, as well as CLD982 12h, 24h, 48h, OS1 24h, and OS2 24h post induction. Assay plate 2 corresponded to: CLD981 OS1 48h and OS2 48h post induction, as well as CLD982 OS1 48h and OS2 48h post induction. Assay plate 3 corresponded to CLD977 and CLD990 for both the second to last and last time point post induction (sonication) as well as the last time point post induction (Bug buster). OS1 contains 20% sucrose. Following preparation of the OS1 fraction, the cell pellet went on to preparation of OS2, which contains MgSC .

Results for assay plate 1 results are shown in Figs. 9 - 13 and Table 2. Specifically, Fig. 9 shows a standard curve of Time (sec) vs. Absorbance for Controls 1 and 2 (combined into control standard) as well as reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L. Controls 1 and 2 were preset dilutions of 1.0 μg/mL ran as duplicates. Fig. 10 shows a standard end point curve of Standard Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec. Specifically, endpoint analysis was carried out in which a reaction was measured at t = 0 and at the end of a specified time interval, and the t = 0 absorbance value was subtracted. For analysis of beta-lactamase, the reaction was measured at time = 60 sec. The absorbance was measured at time = 0 sec which was then subtracted from the 60 sec measurement. Several dilutions of the reference standard were tested to generate a standard curve. Fig. 1 1 shows a standard curve of Time (sec) vs. Absorbance for CLD981 (3/13C037) 12h, 24h, 48h, and OS2 48h post induction. Fig. 12 shows a standard end point curve of Time (sec) vs. Absorbance for CLD981 OS1 samples. Fig. 13 shows a standard curve of Time (sec) vs. Absorbance for CLD982 (4/13C038) 12h, 24h, 48h, and OS1 and OS2 48h post induction. Table 2 shows a summary of assay plate 1 activity and titre results for CLD981 and CLD982 (secretion strains 37 and 38, respectively) along with controls 1 and 2.

Assay plate 2 results are shown in Fig. 14 - 16 and Table 3. Specifically, Fig. 14 shows a standard curve of Time

(sec) vs. Absorbance for Control 1 and 2 (combined into control standard) as well as reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L. Fig. 15 shows a standard end point curve of Standard

Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec. Fig. 16 shows a standard curve of Time (sec) vs. Absorbance for CLD981 (37) and CLD982 (38) OS1 and 0S2 48h post induction. Table 3 shows a summary of assay plate 2 activity and titre results for CLD981 and CLD982 OS1 and OS2 along with controls 1 and 2.

Assay plate 3 results are shown in Fig. 17 - 19 and Table 4. Specifically, Fig. 17 shows a standard curve of Time (sec) vs. Absorbance for Control 1 and 2 (combined as control standard) as well as reference standard material dilutions of 0.6, 0.8, 1.0, 1.5, 2.0, and 4 mg/L. Fig. 18 shows a standard end point curve of Standard Concentration (mg/L) vs. End Point Absorbance. Standard absorbance was measured at time = 60 sec minus standard absorbance at time = 0 sec. Fig. 19 shows a standard curve of Time (sec) vs. Absorbance for CLD977 and CLD 990 (intracellular strains 39 and 40, respectively) for both the second to last and last time point post induction (unlabelled = sonication) as well as the last time point post induction (Bug buster). Table 4 shows a summary of assay plate 3 activity and titre results for CLD977 and CLD990 along with controls 1 and 2.

Tables 2 - 4 specifically show CLD981 , CLD982, CLD977, and CLD990 end point OD, activity concentration (mg/L), assay dilution, concentration x dilution (g/L), whole cell weight (WCW (g/L)), periplasmic dilution factor, g/L P1A activity WB titre, estimated g/L P1A WB by SDS PAGE, SDS PAGE P, and SDS PAGE soluble (if applicable) compared to control 1 and 2.

Table 2. Results from the CENTA method for the periplasmic strains CLD981 (37) and CLD982 (38) at different time points compared to controls 1 and 2.

Table 3. Results from the CENTA method for the periplasmic strains CLD981 (37) and CLD982 (38) at different time points compared to controls 1 and 2. ftetfsiiy

End point Assay e>'LP1 A activity 1 SBS PASE i OE> concentration

iJifatKJB tgitf dilution factor WBtitte CliL VVB

S7- 140S1 ^•3.C42S 0.2S45 417 0.12 0.71 δ.17 &.5S

37- OS2 5.S27S 0 - 2Ϊ.5 1738 8.23 0.71 0.32 1.3

38-1 OS1 0.1SS0 I.03S3 2:38 1 44 ! SS 0.4

S8-140S2 •3.1150 1.1034 12:55 1.3B 1: .44· oss

Assay coni si 1 ::·.: : ·¾ O.S730 32333 3 4

Assay eo tm<! 2 •3.11 5 ^ ¾ 'g^ .32055 31.33

Table 4. Results from the CENTA method for the intracellular strains CLD977 (39) and CLD990 (40) at different time points compared to controls 1 and 2.

End point Activity Assay Activity

OQ concentration concentration X : git. Pt A actw v SBS PAGE

Btfte •SBS PAGE P j soluble dBtiiiorj is

6.1SS0 1.0705 i 20330 41.35 i 41.05 j 11.4 10.9

3SMJ i Sc. nicafcan) £i 1875 1.8583 ; 25000 4S.43 i 4S.48 j 14.5 12.1

40-7 i So nicaiion) 5 1835 1.81S3 I 25000 45.42 4542 11.5 1.3

40-4<Ecnka†!em 3.1SS5 1.S4S7 ; 27780 δΐ,ϊϊ j S1.3S i 13.2 1¾.7

.35-8 <B«s busts) 3.1020 1.0015 i 2S 00 45,53 j 4S.03 ; 14.0 12.1

40-8 (Bus taster* •3.1770 1.7 S5 i 27780 48.68 : 48.68 ; 13.2' 1!3.7

Assay contrai 1 •3.1080 00057 i 32000 31:.8S

Assay confcoi 2 •3.1045 O.S7« j 32000 31. IS

As seen above, for the intracellular strains, there was a marginally greater activity in strain CLD990 compared to strain CLD977. For the periplasmic strains, the best secretion fraction for CLD981 (gene 1 leader) was 0S2 fraction at 1.3 g/L (by SDS-PAGE), whereas the best secretion fraction for CLD982 (gene 7 leader) was SN fraction at -1.0 g/L (which included the non-processed form). Finally, for intracellular strains, it was observed that applying either Bug buster or sonication produced similar activity and SDS-PAGE results for these preparations.

Overall, intracellular activity and SDS PAGE results were more than 10x greater compared to secretion (periplasmic) fractions. This was a surprising result as typically, expressed proteins are collected from the periplasm. Additionally, the intracellular expression yielded β-lactamase in the soluble fraction as opposed to inclusion bodies. Example 5: La e Scale P3A (SYN-004) Production

cGMP manufacturing of P3A (SYN-004) was undertaken. The initial 750-liter cGMP production run used the pAVEway™ platform (FUJIFILM Diosynth Biotechnologies UK). Yields were 5.5 kilograms of >95% pure SYN- 004 active pharmaceutical ingredient (API) drug substance. The GMP manufacturing process was initiated after a successful evaluation that produced high yielding cell lines that exhibited consistent biological activity of P3A (SYN-004). P3A (SYN-004) expression titers were improved by greater than about 15-fold (14 grams of P3A (SYN-004) per liter of E. coli culture broth), compared to the Bacillus platform previously employed for SYN-004's first-generation predecessor (roughly 1 gram of P1A per liter of Bacillus subtillis culture broth, see Example 1). A single chromatography column purification process reproducibly yielded 40-50% P3A (SYN-004) drug substance recovery at purity levels greater than 95%, another marked manufacturing improvement over the previous purification process.

DEFINITIONS

The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.

As used herein, "a," "an," or "the" can mean one or more than one.

Further, the term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language "about 50" covers the range of 45 to 55.

An "effective amount," when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non- limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

Although the open-ended term "comprising," as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of or "consisting essentially of."

As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g. inventive β-lactamases and/or pharmaceutical compositions (and/or additional agents) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. For example, administration of therapeutic agents to a patient suffering from a Gl tract disorder (e.g. CDI) provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. In certain embodiments, the effect will result in a quantifiable change of two-fold, or three- fold, or four-fold, or five-fold, or ten-fold. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder or reduction in toxicity, regardless of whether improvement is realized.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:

1. A method for the production of a beta-lactamase polypeptide in Escherichia coli (E. coli), comprising:

(a) providing a host £ coli cell transformed with a vector comprising a sequence encoding the beta- lactamase polypeptide;

(b) culturing the £ coli cell to induce expression of the beta-lactamase polypeptide in the cytoplasm; and

(c) recovering the beta-lactamase polypeptide from a soluble fraction prepared from the £ coli cell.

2. The method claim 1 , wherein the method yields at least about 5 grams, about 6 grams, about 7 grams, about 8 grams, about 9 grams, or about 10 grams, or about 15 grams of the beta-lactamase polypeptide per liter of culture.

3. The method of claim 1 , wherein the method yields at least about 10 grams of the beta-lactamase polypeptide per liter of culture.

4. The method of claim 1 , wherein the method yields at least about 15 grams of the beta-lactamase polypeptide per liter of culture.

5. The method of any one of the above claims, wherein the £. coli cell is selected from BL21 (DE3) or W3110.

6. The method of any one of the above claims, wherein expression of the beta-lactamase polypeptide in the cytoplasm is induced by adding isopropylthiogalactoside (IPTG) to the culture.

7. The method of any one of the above claims, wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 60% identity with P1 A.

8. The method of any one of the above claims, wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 60% identity with P2A.

9. The method of any one of the above claims, wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 60% identity with P3A.

10. The method of any one of the above claims, wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least 60% identity with P4A.

11. The method of any of the above claims, wherein the beta-lactamase polypeptide comprises an amino acid sequence having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% identity with one of P1A, P2A, P3A and P4A.

12. The method of claim 4, wherein the beta-lactamase polypeptide comprises an amino acid sequence of SEQ ID NO: 5 (P3A).

13. The method of claim 4, wherein the beta-lactamase polypeptide comprises an amino acid sequence of SEQ ID NO: 10 (P2A).

14. The method of any of the above claims, wherein the purity of the beta-lactamase polypeptide is greater than at least about 95%, or about 96%, or about 97%, or about 98%, or about 99%.

15. The method of any of the above claims, wherein the production comprises the use of an expression vector comprising palindromic DNA looping.

16. The method of any of the above claims, wherein the production comprises the use of an expression vector suitable for tightly controlled gene expression.

17. The method of any of the above claims, wherein the production comprises a single chromatography column step.

18. The method of claim 17, wherein the purification process yields about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% beta-lactamase.

19. A method for the production of a beta-lactamase polypeptide in Escherichia coli (E. coli), comprising:

(a) providing a host E. coli cell transformed with a vector comprising a sequence encoding the beta- lactamase polypeptide;

(b) culturing the E. coli cell to induce expression of the beta-lactamase polypeptide in the cytoplasm; and

(c) recovering the beta-lactamase polypeptide from a soluble fraction prepared from the E. coli cell; wherein:

the beta-lactamase polypeptide comprises an amino sequence of SEQ ID NO: 5 (P3A) and the method yields at least about 15 grams of the P3A beta-lactamase polypeptide per liter of culture.